Research Article
Zerumbone attenuates TGF-β1-mediated epithelial-mesenchymal transition via upregulated E-cadherin expression and downregulated Smad2 signaling pathways in
non-small cell lung cancer (A549) cells
You-Cheng Hseua,b, Yu-Chi Huangc, Mallikarjuna Korivic, Jia-Jiuan Wuc, Tzong-Der Wayd, Ting-Tsz Oua, Li-Wen Chiuc, Chuan-Chen Leeb, Meng-Liang Line,*, and Hsin-Ling Yangc,*
aDepartment of Cosmeceutics, College of Pharmacy, China Medical University, Taichung 40402, Taiwan
bDepartment of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan cInstitute of Nutrition, China Medical University, Taichung 40402, Taiwan
dDepartment of Life Sciences, China Medical University, Taichung 40402, Taiwan eDepartment of Medical Laboratory Science and Biotechnology, China Medical University,
Taichung 40402, Taiwan
Running title: Zerumbone attenuates TGF-β1-mediated EMT
*Corresponding authors 1. Dr. Hsin-Ling Yang
Institute of Nutrition, China Medical University 91, Huseh-Shih Road, Taichung 40402, Taiwan
Tel.:＋886 4 22053366 x 7503; fax:＋886 4 22078083. E-mail address: [email protected]
2. Dr. Meng-Liang Lin
Department of Medical Laboratory Science and Biotechnology, China Medical University 91, Huseh-Shih Road, Taichung 40402, Taiwan
ABSTRACT
Zerumbone, a sesquiterpene compound of edible ginger (Zingiber zerumbet), has been tested for
its anti-EMT and anti-metastatic properties in TGF-β1-stimulated human lung cancer (A549)
cells. Zerumbone (10/20 µM) treatment prior to TGF-β1-stimulation reversed the adverse
morphological changes (fibroblastic-to-epithelial phenotype), and up-regulated E-cadherin
expression against TGF-β1-induced down-regulation. Immunofluorescence and luciferase
activity data confirmed the up-regulated E-cadherin expression and transcriptional activity by
zerumbone under β1-stimulation. Further evidence showed that zerumbone decreased
TGF-β1-mediated phosphorylation and transcriptional activity of Smad2, but not Smad3. These results
revealed zerumbone inhibit the TGF-β-induced EMT via up-regulation of E-cadherin and
down-regulation of Smad2 signaling pathways. Findings from wound-healing, invasion and colony
formation experiments convinced that zerumbone inhibits TGF-β1-mediated (metastatic)
migration, invasion and anchorage-independent growth. Besides, zerumbone alone is capable to
induce autophagy and apoptosis in A549 cells. These results conclude that EMT and
anti-metastatic activities of zerumbone may contribute to develop food-based chemopreventive drugs
for non-small cell lung cancer treatment.
1. Introduction
Lung cancer is the major cause of cancer-related mortality among both men and women around the World. Non-small cell lung cancer (NSCLC) is the most common form of lung cancer accounting for approximately 80% of all cases (Tsim, O'Dowd, Milroy, & Davidson, 2010). The prognosis of NSCLC is still very poor, with the 5-year survival rate of <20% (Esposito, Conti, Ailavajhala, Khalil, & Giordano, 2010; Wang, Nelson, Bogardus, & Grannis, 2010). Advanced treatments, such as surgery and chemotherapy provide only limited improvement in the survival of patients with NSCLC. A continuing problem in the management of NSCLC is tumour metastasis, which is limiting the treatment benefits and increasing numbers of cases (Zarogoulidis et al., 2013). This context urging the importance of further in-depth studies to understand the biochemical changes occurs in tumour cells to promote the aggressive neoplastic phenotype.
Epithelial-mesenchymal transition (EMT) is an important process in cancer cells, where fully developed epithelial cells undergo migration, invasion and metastasis (Willis & Borok, 2007). The loss of cell-cell adhesion, reorganization of the actin cytoskeleton and acquisition of increased migratory characteristics are the hallmarks of EMT. Tumour invasion represents the first and essential step in the metastatic cascade of carcinomas that requires profound changes in cell adhesion, and enables tumour cells to dissociate and migrate from the primary site (Thiery, Acloque, Huang, & Nieto, 2009). Transforming growth factor-β1 (TGF-β1), a multifunctional cytokine can induce EMT during wound healing, embryonic development, fibrotic diseases and cancer pathogenesis (Massague, Blain, & Lo, 2000; Scanlon, Van Tubergen, Inglehart, & D'Silva, 2013). EMT is fundamental during transition from epithelial to mesenchymal myofibroblast-like cells accompanied by a decreased expression of E-cadherin (Thiery, 2002;
Willis & Borok, 2007). E-cadherin is an adherent junction protein, specifically expressed in epithelial cells, and loss of this cell surface protein is a universal feature of EMT (Arias, 2001). Several signaling pathways have been implicated in TGF-β1-induced EMT in cancer cells, including Smad-dependent signaling cascades. The phosphorylated Smad2 and Smad3 proteins with Smad4 subsequently translocate into the nucleus, where they regulate the transcription of EMT-associated genes (Ikushima & Miyazono, 2010). However, suppression of TGF-β1-induced EMT, and responsible molecular events involved in EMT inhibition under anticancer treatment has not been investigated in lung cancer cells.
Ginger rhizome and its extracts have been used as a spice in preparation of functional foods since ancient times around the world. Recent studies emphasized that key components in functional foods, including ginger could promote health and combat against several diseases (Embuscado, 2015; Stoilova et al., 2007). In Southeast Asia, rhizomes of edible ginger, Zingiber zerumbet (Zingiberaceae) have been used as a traditional medicine to treat stomachache, toothache, fever and indigestion. Zerumbone is a sesquiterpene phytochemical (Figure 1A) naturally occurring from the rhizomes of Z. zerumbet (Murakami et al., 2002; Yob et al., 2011). Some laboratory in vitro and in vivo studies indicated the proliferative, antioxidant, anti-inflammatory and anticancer properties of zerumbone in a variety of cancer models, but no or less effect on normal cells (Kapoor, 2012; Murakami et al., 2002; Prasannan et al., 2012). Recently zerumbone-induced mitochondrial apoptosis was reported in NSCLC (A549) cells, which was evidenced by loss of mitochondria membrane potential, increased release of cytochrome-c from mitochondria, and activation of caspase-9 and caspase-3 (Hu, Zeng, Zhang, Liu, & Wang, 2014). Despite its well-known anticancer properties, relatively little is known about the inhibitory effects of zerumbone on lung EMT and metastasis, and underlying molecular mechanisms responsible for its therapeutic effects are largely unknown.
In this study, we developed a well-validated model of EMT in A549 human NSCLC cell lines, and explored the zerumbone capabilities in inhibiting the TGF-β1-induced EMT and associated changes. We used A549 cell lines, because A549 cells possess many features of normal alveolar epithelial cells, and have been used in numerous studies to examine the TGF-β1-induced EMT (Kasai, Allen, Mason, Kamimura, & Zhang, 2005; Ranganathan et al., 2007). The levels of EMT/metastatic control and involved key molecular biomarkers were assayed to determine the zerumbone-mediated anti-EMT and anti-metastatic properties, and attempted to explain the underlying mechanism in A549 cells under TGF-β1-stimulation.
2. Materials and methods
2.1. Reagents and Antibodies
Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin were obtained from GIBCO BRL/Invitrogen (Carlsbad, CA, USA). Zerumbone and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against E-cadherin, phospho-Smad2, Smad2, phospho-Smad3, Smad3, LC3-I/II, p62, caspase-3, and PARP were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against β-actin were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). All other chemicals were purchased from either Merck & Co., Inc. (Darmstadt, Germany) or Sigma-Aldrich.
2.2. Cell culture
The human lung cancer A549 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were grown in DMEM/F12 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO2. Cultures were harvested and cell morphology was examined using phase
contrast microscopy. Cell number was monitored by counting cell suspensions using a hemocytometer.
2.3. Drug treatment
For all TGF-β1-stimulated experiments, the supernatant was removed after zerumbone supplementation for 2 h, the cells were washed with PBS and the culture medium was replaced with new medium, and then stimulated with or without TGF-β1 (2 ng/mL) for 24 h. In this study, 2 h zerumbone pretreatment was determined based on the results from the time-dependent studies of zerumbone on A549 cell viability. MTT data showed that 20 μM zerumbone had no any adverse effects on A549 cell survival at 2 h after treatment. Therefore, 2 h zerumbone (20 μM) treatment was considered as optimum time-point on A549 cell viability, and continued further studies.
2.4. MTT assay
Cell viability was determined by the MTT colorimetric assay. Cells (5 × 104 cells/well in 24-well plates) were treated with zerumbone alone (10-80 μM for 2 h), or pre-treated with zerumbone (10 or 20 μM) for 2 h, and then stimulated with or without TGF-β1 (2 ng/mL) for 24 h. MTT (0.5 mg/mL) in PBS was added to each well. After incubation at 37 °C for 4 h, an equal volume of 90% isopropanol and 0.5% sodium dodecylsulphate (SDS) mixture (400 μL) was added to dissolve the MTT formazan crystals, and the absorbance was measured at 570 nm (A570) using an ELISA microplate reader (µ-Quant, Winoosky, VT, USA). The percentage (%) of cell viability was calculated as: (A570 of treated cells/A570 of untreated cells) × 100.
2.5. In vitro wound-healing repair assay
To assess cell migration, A549 cells were seeded into a 12-well culture dish and grown in DMEM/F12 containing 10% FBS to a nearly confluent cell monolayer. The cells were re-suspended in DMEM/F12 medium containing 1% FBS, and a “wound gap” in the monolayers
were carefully scratched using a culture insert. Cellular debris was removed by washing with PBS, and then the cells were incubated with a non-cytotoxic concentration of zerumbone (10 or 20 μM) for 2 h and washed. Then cells were stimulated with or without TGF-β (2 ng/mL) for 24 or 48 h. The migrated cells were photographed (100 × magnification) at 0, 24 and 48 h to monitor the migration of cells into the wounded area, and the closure of the wounded area was calculated.
2.6. Cell invasion assay
Invasion assays were performed using BD Matrigel invasion chambers (Bedford, MA, USA). For the invasion assay, 10 µL Matrigel (25 mg/50 mL) was applied to 8-µm polycarbonate membrane filters, 1 × 105 cells were seeded to the matrigel-coated filters in 200 µL of serum-free medium containing zerumbone (10 or 20 μM) and/or TGF-β1 (2 ng/mL). The bottom chamber of the apparatus contained 750 µL of complete growth medium. Cells were allowed to migrate for 24 h at 37 °C. After 24 h incubation, the non-migrated cells on the top surface of the membrane were removed with a cotton swab. The migrated cells on the bottom side of the membrane were fixed in cold 75% methanol for 15 min and washed 3 times with PBS. The cells were stained with Giemsa stain solution and then de-stained with PBS. Images were obtained using an optical microscope (200 × magnification), and invading cells were quantified by manual counting.
2.7. Protein isolation and Western blot analysis
A549 cells were seeded in a 10 cm dish at a density of 1 × 106 cells/dish. Next, the cells were incubated with zerumbone (10 or 20 μM) for 2 h, then stimulated with or without TGF-β1 (2 ng/mL) for 48 h. Cells were detached and washed once in ice-cold PBS, and then re-suspended in 100 L lysis buffer containing 10 mM Tris-HCl [pH 8], 0.32 M sucrose, 1% Triton X-100, 5 mM EDTA (ethylenediaminetetraacetic acid), 2 mM dithiothreitol, and 1 mM phenylmethyl sulphonyl fluoride. The suspension was kept on ice for 20 min, and then centrifuged at 15,000 × g for 30 min at 4 °C. Total protein content was determined using the Bio-Rad protein assay
reagent (Bio-Rad, Hercules, CA, USA), with bovine serum albumin (BSA) as standard. Protein extracts were reconstituted in sample buffer (0.062 M Tris-HCl, 2% SDS, 10% glycerol, and 5% β-mercaptoethanol), and the mixture was boiled for 5 min. Equal amounts (50 g) of the denatured proteins were loaded onto each lane, separated on 8-15% SDS polyacrylamide gels, followed by transfer of the proteins to polyvinylidene difluoride membranes overnight. Membranes were blocked with 0.1% Tween-20 in PBS containing 5% non-fat dried milk for 20 min at room temperature, and the membranes were reacted with primary antibodies for overnight. The membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody for 2 h. The blots were detected using ImageQuant™ LAS 4000 mini (Fujifilm, Tokyo, Japan) with Enhanced Chemiluminescence substrate (Millipore, Billerica, MA, USA). Densitometry analyses were performed using commercially available quantitative software (AlphaEase, Genetic Technology Inc. Miami, FL, USA) with the control representing 1.0-fold as shown below the data.
2.8. Luciferase activity assay of E-cadherin or Smads
The E-cadherin or Smads transcriptional activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI, USA). A549 cells were cultured in 24-well plates that had reached 70–80% confluence, incubated for 5 h with serum-free DMEM/F12 that did not contain antibiotics. The cells were then transfected with either a pcDNA vector or an E-cadherin/Smads (3TP-Luc reporter vector) plasmid with β-galactosidase and GFP using Lipofectamine 2000 (Invitrogen). After plasmid transfection, cells were pre-treated with zerumbone (10 or 20 μM) for 2 h, and then stimulated with or without TGF-β1 (2 ng/mL) for 24 h. After treatment, the cells were lysed and their luciferase activity was measured using a luminometer (Bio-Tek instruments Inc, Winooski, VA, USA). The luciferase activity was normalized to the β-galactosidase activity in cell lysate and considered basal level (100%). The luciferase activity was normalized to the β-galactosidase activity in the cell lysates, and the data were expressed as the averages of three independent experiments.
2.9. Fluorescent imaging of E-cadherin
A549 cells (2 × 104 cells/well) were seeded onto an eight-well glass Tek chamber and pre-treated with zerumbone (10 or 20 μM) for 2 h, then stimulated with or without TGF-β1 (2 ng/mL) for 24 h. After treatment, cells were fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and then incubated for 1 h with anti-E-cadherin primary antibodies in 1.5% FBS. The cells were then incubated with FITC (fluorescein isothiocyanate)-conjugated (488 nm) secondary antibodies for an additional 1 h in 6% bovine serum albumin. The cells were then stained with 1 μg/mL DAPI for 5 min. The stained cells were washed with PBS and visualized using a fluorescence microscope at 200 × magnification.
2.10. Colonyformation assay
Anchorage-independent growth was determined by colony formation using the soft agar method. The assay was performed in 6-well plates (2 × 105 cells/well in 3-cm dishes) with a base layer containing 0.5% agar in DMEM/F12 containing 10% FBS, 1 mM glutamine, and 100 units of penicillin plus 100 g/mL of streptomycin. This layer was overlaid with a second layer of 1 mL of 0.35% agar (in DMEM/F12 containing 10% FBS, 1 mM glutamine, and 100 units of penicillin plus 100 g of streptomycin) with a suspension of 1 × 104 cells/well. Fresh medium with zerumbone (10 or 20 μM) for 2 h and/or TGF-β1 (2 ng/mL) then added to the plates for 24 h. The plates were incubated at 37 °C for 7 days, and the tumour colonies were determined with a microscope. The numbers of colonies >200 µm in size were counted using an electron microscope (40 × magnification). Colonies were subsequently stained with p-iodonitrotetrazolium violet (1 mg/mL), and colonies larger than 200 µm were counted. The percentage of colony formation was calculated by defining the number of colonies in the absence of zerumbone as 100%.
2.11. Statistical analyses
using analysis of variance followed by Dunnett’s test for pair-wise comparison. An asterisk indicates that the experimental values were significantly different from those of the controls (*P < 0.05) and # indicates values are significant compared to TGF-β1 alone treated cells.
3. Results
3.1. Effect of zerumbone on cell viability/proliferation of non-small cell lung cancer (A549) cells
The cytotoxic effects of zerumbone (Fig. 1A) on non-small cell lung cancer (A549) cells were examined prior to investigating the anti-EMT and anti-metastatic properties in the presence or absence of TGF-β1. Result showed that A549 cell proliferation was greatly increased by TGF-β1 from 100% to 142% as increases the concentration (1-20 ng/mL, Fig. 1B). Zerumbone alone treatment for 2 h didn’t affect the cell viability from low to high concentration (10-80 µM, Fig. 1C). Interestingly, increased cell viability with TGF-β1 stimulation (2 ng/mL) for 24 h was slightly inhibited by 2 h zerumbone pretreatment (10-20 µM, Fig.1D). Based on this data, the non-cytotoxic concentrations of zerumbone (10 or 20 µM) with 2 h pretreatment was used to evaluate its anti-EMT and anti-metastatic properties in TGF-β-stimulated A549 cells.
3.2. Zerumbone suppressed TGF-β-induced cell scattering and EMT in A549 cells
A549 cells were treated with zerumbone (10 or 20 µM, 2 h) prior to TGF-β1 stimulation (2 ng/mL) for 24 h, and then zerumbone effect on TGF-β1-induced cell scattering and EMT was determined under phase-contrast microscope. We found a classic cobblestone epithelial morphology and growth pattern of cells that were cultured without TGF-β1. However, in the presence of TGF-β1, cells adopted a fibroblast-like morphology and reduced their cell-cell contact (Fig. 2A). Here it is important to note that the altered morphology was not observed in zerumbone pretreated cells under TGF-β1-stimulation; instead, zerumbone retained the cobblestone epithelial morphology. We speculate that TGF-β1 stimulation may propagate the EMT process in cancer cells, which could be attenuate by zerumbone treatment.
3.3. Zerumbone suppress the TGF-β1-induced EMT via restoration of E-cadherin
As we expected, the abolishment of cell scattering by zerumbone is associated with the management of EMT against TGF-β1-induced dysregulation. To confirm this phenomenon, E-cadherin, an epithelial phenotype protein involved in regulation of EMT was assayed in the cells. Our findings confirmed that TGF-β1 stimulation downregulated the E-cadherin expression in cancer cells, which is a hallmark of EMT. Nevertheless, zerumbone pretreatment attenuated the TGF-β1-induced downregulation of E-cadherin, and the restored E-cadherin protein was significant with 20 µM of zerumbone compared to TGF-β1 alone (Fig. 2B).
The increased E-cadherin by zerumbone may be due to the activation of E-cadherin transcriptional activity. Therefore, E-cadherin promoter activity was measured using luciferase activity, and the data was presented in Fig. 2C. In this study, E-cadherin promoter construct in a pcDNA vector was transiently transfected into A549 cells, and luciferase activity was measured. Results showed that luciferase activity derived from the E-cadherin promoter was consistently increased with zerumbone (10-20 µM) in a dose-dependent manner against TGF-β1-induced suppression (Fig. 2C). The luciferase activity of blank plasmid pcDNA in A549 cells was not affected by zerumbone (data not shown). Furthermore, either E-cadherin protein expression or transcriptional activity was not affected by zerumbone alone (20 µg/mL) (Fig. 2A-C).
Further evidence of E-cadherin down-regulation by TGF-β1 and EMT was obtained from the immunofluorescence assay. As illustrated in Fig. 3A and B, immunofluorescence staining using E-cadherin antibodies showed a down-regulation of E-cadherin in TGF-β1 treated cells. However, pretreatment with zerumbone for 2 h significantly suppressed such down-regulation of cadherin in a dose-dependent manner. Since EMT is characterized by down-regulation of E-cadherin at molecular level (Yuki, Yoshida, Inagaki, Hiai, & Noda, 2014), our results explain that zerumbone could inhibit the TGF-β1-induced EMT via up-regulation of E-cadherin transcriptional activation.
3.4. Zerumbone attenuates TGF-β1-induced EMT through the inhibition of Smad2 signaling pathways in A549 cells
TGF-β (by acting on TβRI) was reported as a major secretory ligand stimulating Smads activation and induces Smad2 phosphorylation at Ser465/467 (Derynck & Zhang, 2003). A549 cells were treated with zerumbone (20 μM for 2 h) prior to TGF-β1 (2 ng/mL) stimulation for 24 h, and then stained with respective (Smad2 and Smad3) antibodies. TGF-β-induced Ser465/467-phosphorylation of Smad2 was observed within 0.25 h of stimulation, which reached a maximum between 0.5 and 1 h after stimulation, and then started to decline within 24 h of the experimental period without affecting the total Smad2 expression (Fig. 4A). Interestingly, zerumbone pretreatment suppressed the TGF-β-induced excessive Ser465/467-phosphorylation of Smad2. Nonetheless, TGF-β-induced Ser423/425-phosphorylation of Smad3 was unaffected by zerumbone, and no significant changes were observed in total proteins of Smad2 and Smad3 (Fig. 4A).
Next we examined the Smad-dependent transcription with a luciferase reporter construct that was stably transfected into A549 cells. Luciferase reporter assay showed that TGF-β1 (2 ng/mL) stimulation for 24 h caused a profound increase in Smads (3TP-Luc) transcriptional activity. In consisting with phosphorylation, zerumbone (10-20 μM, 2 h) significantly decreased the Smads transcriptional activity in a dose-dependent manner, which is almost close to the control cells (Fig. 4B). These findings confirmed that zerumbone can attenuate the TGF-β-activated EMT through inhibition of Smad2 signaling pathways in A549 cells.
3.5. Zerumbone pretreatment inhibits TGF-β1-induced migration of A549 cells
Cancer metastasis, spreading of tumour cells from the primary neoplasm to distant sites and their growth is the most common cause of death in cancer patients (Yang & Weinberg, 2008). Hence, inhibition of migration/invasion could be a potential strategy to prevent or inhibit cancer metastasis (Bjorklund & Koivunen, 2005). A classical wound-healing in vitro assay was
performed to determine the anti-migration property of zerumbone under TGF-β1 stimulation. For this assay, confluent monolayers of A549 cells were incubated with or without zerumbone (10 or 20 µM) for 2 h in the presence or absence of TGF-β1 (2 ng/mL) stimulation for 24 or 48 h.
As shown in Fig. 5A and B, identical evidence demonstrated that exposure to TGF-β1 alone significantly (p < 0.05) induced the migration of A549 cells after 24 or 48 h, whereas zerumbone pretreatment significantly (p < 0.05) prevent the TGF-β-induced tumour cell migration in a dose-dependent manner. Despite its anti-migration property under TGF-β1 stimulation, zerumbone alone (20 µM) treatment for 2 h did not affect the endogenous migratory potential of A549 cells assayed at 24 and 48 h after treatment, which is almost similar to untreated cells.
3.6. Zerumbone pretreatment inhibits TGF-β1-induced invasion of A549 cells
Invasion, the ability of cells to pass through a layer of extracellular matrix on a Matrigel-coated filter was determined in zerumbone pretreated cells under TGF-β1 exposure (2 ng/mL). The photographed invasion of cancer cells were presented in Fig. 6A and B. We found that TGF-β1 greatly induced the invasiveness of A549 cells 24 h after stimulation compared to untreated cells. Consistent with the anti-migration property, TGF-β1-induced invasion was substantially (p < 0.05) inhibited with 10 and 20 µM of zerumbone pretreatment. However, the invasive potential of A549 cells was not affected by zerumbone (20 µg/mL) alone (Fig. 6A and B).
3.7. Zerumbone inhibits tumourigenic ability of A549 cells against TGF-β1 induction
The colony formation ability of cancer cells is directly proportionate to the aggressive potential of the specific cell line. We next evaluated the ability of TGF-β-induced aggressive A549 cells to form colonies on 6-well culture plates in the presence or absence of zerumbone for 7 days. The observed growth of tumour cells in soft agar was presented in Fig. 7A and B. Results showed that TGF-β1 exposed A549 cells produced many colonies compared to untreated cells, while colony numbers were significantly (p < 0.05) suppressed by 10 and 20 µM zerumbone. The
suppressed colony formation ability was more prominent with 20 µM zerumbone. However, the same concentration of zerumbone (20 µM) alone did not influence the colony formation ability of A549 cells. The decreased colony number or colony formation ability of non-small cell lung cancer A549 cells with zerumbone implies the decreased rate of tumourigenic ability.
3.8. Various concentrations of zerumbone alone induce autophagy and apoptosis in A549 cells
Our initial findings showed that zerumbone had no adverse effects on cell viability with 10 to 80 µM concentration after 2 h incubation. In continuation with this, we extended the incubation period to 24, 48 and 72 h, and the effect of zerumbone (10-50 µM) on viability of A549 cells was tested. We found that zerumbone incubation for 24, 48 and 72 h induced a significant diminution of A549 cell viability in a dose-dependent manner with IC50 values of 494, 44 and 21 μM at respective time-points. This data indicates the zerumbone abilities to impair the tumour growth and destruct the cells when tumour cells exposed to longer period (Fig. 8A). Our results further suggest that zerumbone alone could suppress the growth and survival of A549 cancer cells.
To evaluate the underlying mechanism behind zerumbone-mediated cell death, A549 cells were incubated for 48 h with zerumbone (10-30 µM), and key proteins involved in cell death, including LC3I/II, p62, caspase-3, and PARP were determined by Western blot. As shown in Fig. 8B, we found a significant increase of LC3-I/II expression in zerumbone treated A549 cells, which indicates 48 h incubation could cause autophagy of tumour cells in a concentration dependent manner. Another protein, p62 serves as a marker for the induction of autophagy has been found to increase with 48 h incubation of zerumbone.
In connection with zerumbone autophagy property, next we monitored the apoptosis marker proteins under same experimental conditions. Western blot analyses showed that incubation of A549 cells with zerumbone induced the proteolytic cleavage of procaspase-3 (35 kDa) into their active forms (17 kDa) (Fig. 8C). PARP-specific proteolytic cleavage by caspase-3 is considered
to be a biochemical characteristic of apoptosis. Fig. 8C also showed that the 116 kDa PARP protein cleaved into a 89 kDa fragment in response to zerumbone in A549 cells. Taken together, our findings suggest that zerumbone may induce both autophagy and apoptosis in A549 cells, which was accompanied by the LC3-I/II conversion, p62 expression, caspase-3 activation and PARP degradation.
4. Discussion
Ginger ingredients, key components in functional foods have been used for centuries, and reported to promote health and combat against several chronic diseases. In this study, we demonstrated the anti-EMT and anti-metastatic capabilities of zerumbone, and provided possible mechanisms responsible for its effects in A549 cancer cell lines under TGF-β1 stimulation. We used A549 cancer cells as a model of human non-small cell lung cancer (NSCLC), and converted them into fibroblastic phenotype by exposing to TGF-β1. The convincing evidence indicated that TGF-β1 stimulation caused loss of cells’ polygonal appearance, cell-cell contacts, and then leads to acquisition of elongated, spindle shaped morphology consistent with fibroblasts. Furthermore, down-regulation of E-cadherin and changes in EMT-linked signaling regulators suggest that A549 cells are highly sensitive to TGF-β1 that could initiate and propagate the EMT process in cancer cells. The beneficial effect of zerumbone pretreatment was evidenced by restoration of cadherin protein and transcriptional activity against TGF-β1-induced loss. The restored E-cadherin was associated with the inhibition of Smad2 phosphorylation, which is a key molecular event in the suppression of TGF-β1-induced EMT. In convincing to these effects, increased cancer cell migration, invasion and colony formation ability by TGF-β1 were effectively suppressed by zerumbone pretreatment. These findings suggest that zerumbone acts as anti-EMT and anti-metastatic substance, and explain the possible molecular signaling pathways involved in this phenomenon.
Ginger has been an important ingredient in Chinese and Indian herbal medicine apart from its culinary use as functional food. The chemical components in ginger root have shown potent antioxidant, antidiabetic and antilipidemic properties under various stress conditions. The combination of nutritional and medicinal benefits can determine ginger extract as a functional food (Embuscado, 2015; Mallikarjuna, Chetan, Reddy & Rajendra, 2008; Stoilova et al., 2007). In this context, Zerumbone, a naturally occurring cyclic sesquiterpene isolated from the rhizomes of dietary ginger Zingiber zerumbet, has been shown to inhibit the TGF-β1-induced A549 cell scattering and morphological destruction. The TGF-β1-induced cell disruption was characterized by the loss of classic cobblestone epithelial morphology, reduced cell-cell contacts, and acquired mesenchymal spindle-like fibroblast appearance. A profound decrease in E-cadherin expression along with morphological alterations is a universal feature of EMT process in the presence of TGF-β1. Thus, our findings affirmed the successful establishment of EMT model in A549 cells to study the therapeutic effects of zerumbone on the consequences of cell pathology.
It has been demonstrated that TGF-β can enhance the tumour progression by stimulating the complex process of EMT. The markers of TGF-β-induced EMT were indicated by the acquisition of stress fibres, fibroblastic morphology, loss of junctional E-cadherin localization, and increased cellular motility (Bhowmick et al., 2001). TGF-β regulates at least two components of EMT progression, including regulation of actin cytoskeleton and rearrangement of adherens junctions, however, the predominant pathway involved in TGF-β-mediated EMT appears to be highly cell type and context dependent (Willis & Borok, 2007; Bhowmick et al., 2001). Reversal or inhibition of EMT could manage the tumour progression and alleviate the cancer pathology. Zerumbone pretreatment in our study effectively suppressed the TGF-β1-induced EMT in lung cancer cells, as indicated by cobblestone epithelial morphology. Because actin stress fibres are important for cell motility, disruption of actin stress fibres by zerumbone may account for the inhibition of TGF-β1-induced scattering and motility, however this phenomenon remains to be confirmed. A previous study demonstrated that hepatic growth factor
blocks TGF-β1-stimulated EMT in human kidney epithelial cells apparently by a mechanism independent of Smads phosphorylation and their subsequent nuclear translocation (Yang, Dai, & Liu, 2005). Therefore, understanding of precise molecular mechanism mediating TGF-β1-induced EMT in A549 cells, and interactions of zerumbone with other signaling pathways could be essential for developing strategies to inhibit EMT without affecting the beneficial effects of TGF-β1 signaling.
E-cadherin is an adherent junction protein that is specifically expressed in epithelial cells (Kudo et al., 2004). In this study, we determined the protein levels and transcriptional activity of E-cadherin to define the occurrence of EMT with TGF-β1-stimulattion in lung cancer cells. Western blot and Immunofluorescence/luciferase activity data reveals that TGF-β1 is capable of destabilizing E-cadherin junctions by regulating actin organization in A549 cells, which is consistent with previous reports (Bhowmick et al., 2001). The loss of E-cadherin promotes EMT that is responsible in the progression of cancer cells to metastatic state. Although the precise mechanism responsible for E-cadherin inactivation is unclear in cancer cells, alterations at transcriptional levels, appear to be one of the possible reasons for its down-regulation (Chu et al., 2006; Peinado, Portillo, & Cano, 2004; Thiery, 2002). Thus, restoration or prevention of E-cadherin down-regulation could be a useful strategy to control the progression of metastasis and EMT. The restored E-cadherin transcriptional activity and protein level with zerumbone in our study has shown to inhibit the EMT and cancer cell metastasis. Since reduced E-cadherin is due to the increased methylation of its promoter region (Kudo et al., 2004), we assumed that restored E-cadherin by zerumbone possibly mediated through the demethylation of E-cadherin promoters. Further evidences emphasized that zerumbone increased the luciferase activity derived from E-cadherin promoter, which might be a responsible signaling pathway involved in suppression of EMT. This phenomenon may explain the interference of zerumbone on the cross-talk between E-cadherin and EMT cascades. However, response of other signaling pathways, including receptor tyrosine kinases (RTKs) with zerumbone on the inhibition of E-cadherin and EMT remains to be
studied. Restoration of E-cadherin by garlic-derived compounds has been associated with the inhibition of proliferation and morphological changes in prostate cancer cells (Chu et al., 2006).
For the first time, here we provided the possible signaling pathways responsible for the restoration of E-cadherin and inhibition of EMT with zerumbone in lung cancer cells. Smads, the transcription factors constantly shuttles between cytoplasm and nucleus play a crucial role in mediating the intracellular response to TGF-β (Zhang, 2009). TGF-β-mediated Smad signaling in EMT associated with tumour progress and development, with differential involvement of Smad2 or Smad3, depending on cellular context (Willis & Borok, 2007). In lung A549 cancer cells, Smad2 signaling has been shown to play a key role in the induction of TGF-β-mediated EMT (Kasai et al., 2005). Consisting with the previous reports, our findings demonstrated the essential role of Smad2 signaling in TGF-β1-induced EMT in A549 cell lines. During Smad-dependent EMT, TGF-β1 signals are transduced by transmembrane serine/threonine kinase type II and type I receptors. Upon TGF-β1 stimulation, the receptors are internalized into early endosomes, where Smad anchor for receptor activation (SARA) modulates the formation of complexes with Smad2 or Smad3. Then Smad2 and Smad3 phosphorylated at serine residues by the type I receptor. Phosphorylation induces their association with Smad4 and translocation to the nucleus, and mediates gene transcription by binding to Smad binding elements in the promoters of its target genes (Derynck & Zhang, 2003; Willis & Borok, 2007). Increased Smads transcriptional activity (p3TP-Luc) with TGF-β1 stimulation has been found to decrease in zerumbone treated cells. These findings imply the Smad-dependent EMT in lung A549 cancer cell lines that could be handled by zerumbone; but Smad-independent pathway remains to be investigated.
Our experimental results revealed that zerumbone pretreatment effectively attenuated the TGF-β1-induced Smad2 phosphorylation and transcriptional activity in A549 cells. This phenomenon was accompanied by a substantial restoration of E-cadherin levels and suppressed EMT in A549 cells. Blocking of TGF-β-induced Smad2 phosphorylation, nuclear accumulation, and target gene expression by a specific inhibitor of TGF-β1 (GW788388) has been found to
decrease the EMT and renal fibrosis (Petersen et al., 2007). In this context, zerumbone could act as an inhibitor of TGF-β1-mediated signaling cascades. Thus, our findings emphasized that inhibition of TGF-β1-induced EMT by zerumbone is mediated by the regulation of Smad2 signaling pathways in lung cancer cells. TGF-β-induces EMT in A549 cells through two main signaling pathways, including Smad-dependent and Smad-independent. Literature reveled that both Smad-dependent and Smad-independent pathways takes place in the induction of EMT, and these signaling cascades may differ based on the cell type and experimental condition (Derynck & Zhang, 2003; Willis & Borok, 2007; Zhang, 2009). In this study, we explored only dependent pathway, therefore, the question of whether zerumbone attenuated EMT via Smad-independent pathway remains to be investigated. Nevertheless, other studies indicated that Smad-independent pathways are much less important compared to the Smad-dependent pathways, because Smad-independent pathways may not sufficient for induction of full EMT. The Smad-dependent pathway is both sufficient and indispensable for EMT (Yang, Chen & Sun ,2013; Zavadil & Bottinger, 2005).
In this study, zerumbone pretreatment not only restored the E-cadherin expression (suppress EMT), but also inhibited cancer cell migration, invasion and colony formation ability against TGF-β1-stimulation. Prolonged exposure of tumour cells to inflammatory cytokines has been shown to induce EMT, which is the principal mechanism involved in metastasis and tumour invasion (Thiery, 2002). Here we found TGF-β1 exposure increased metastasis, invasion, and migration of A549 cells as a consequence cytokine-induced EMT. The occurrence of EMT in A549 cells permits tumour cells to leave the primary environment and migrate as circulating tumour cells to distant sites. The polarized epithelial cells then transformed into highly migratory fibroblastoid cells, lose their polarity, epithelial markers, and cell–cell contact, and thereby enhanced cell motility and invasiveness (Scanlon et al., 2013). Our in vitro wound-healing and invasion studies showed that zerumbone could inhibit the TGF-β-induced metastatic migration and invasion of NSCLC A549 cells. Metastatic inhibitory properties of zerumbone were strongly
supported by its anti-EMT properties in lung cancer cell lines. Water-soluble compounds of garlic were able to inhibit the prostate cancer cell proliferation and invasive abilities, which was associated with restoration of E-cadherin expression (Chu et al., 2006). A recent study indicated that zerumbone can suppress the IL-β-induced cell migration and invasion in human triple-negative breast cancer cells (Han et al., 2014). Furthermore, zerumbone has been shown to suppress the chemokine receptor CXCR4 expression that is correlated with the inhibition of CXCL12-induced invasion of both breast and pancreatic cancer cells (Sung et al., 2008). Cancer metastasis, the spread of tumour from primary site to distance site and growth is the most common cause of death. Zerumbone possesses anti-proliferative properties towards several cancer cell lines with minimal or no effect on normal cells (Kapoor, 2012; Murakami et al., 2002; Rahman et al., 2013; Sadhu, Khatun, Ohtsuki, & Ishibashi, 2007). Thus inhibition of cancer cell migration, invasion, and colony formation abilities of zerumbone could prevent the cancer metastasis and avoid the lethal effects.
Our findings further demonstrated that zerumbone is capable to induce autophagy and apoptosis and in NSCLC A549 cells. Autophagy is a lysosomal degradation pathway that functions in both cell survival and cell death. The role of autophagy in cancer is complex and controversial (Levine, 2007). Several studies have shown a cancer promoting role for autophagy, since the pathway can function to sustain viability during nutrient limitation, growth factor deprivation, and metabolic stress. In contrast, other evidence suggests that autophagy may have an anticancer role. p62 is a receptor for cargo destined to be degraded by autophagy, including ubiquitinated protein aggregates destined for clearance. The p62 protein is able to bind ubiquitin and also to LC3, thereby targeting the autophagosome and facilitating clearance of ubiquitinated proteins (Pankiv et al., 2007). Increased p62 expression with zerumbone may explain that zerumbone could induce autophagy in NSCLC A549 cells. Furthermore, LC3-I/II, widely used marker proteins for autophagy have been found to augment with zerumbone. The estimated apoptotic proteins in A549 have been found to up-regulate with zerumbone treatment (48 h),
which confirmed its pro-apoptotic properties. Similar to our findings, zerumbone (50 µM) has been shown to inhibit proliferation and promote apoptosis in human colonic adenocarcinoma cell lines. It is emphasized that α,β-unsaturated carbonyl group in zerumbone may play a pivotal role in interaction with unidentified target molecules (Murakami et al., 2002). An in vivo study by Kim et al., (2009) reported that zerumbone can suppress lung carcinogenesis in ICR mice (Kim et al., 2009). Another recent study indicated zerumbone induced apoptosis in A549 cells as evidenced by loss of mitochondrial membrane potential, increased release of cytochrome c, and activation of caspase-9 and caspase-3 (Hu et al., 2014). Based on its potent autophagy and apoptosis properties zerumbone could serve as anticancer substance to treat the lung cancer.
For the first time, our findings demonstrated the anti-EMT and anti-metastatic properties of zerumbone in A549 lung cancer cells under stimulatioon. The inhibition of TGF-β1-induced EMT in zerumbone pretreated cells was associated with the restoration of E-cadherin protein and transcriptional activity. Inhibition of TGF-β1-induced Smad2 phosphorylation and transcriptional activity by zerumbone may contribute for a substantial restoration of E-cadherin, thereby suppressed the EMT. Furthermore, suppressed EMT by zerumbone was accompanied by a suppressed cancer cell migration, invasion, and colony formation abilities. These findings provided novel insights in understanding the possible molecular mechanisms underlying the promising anticancer properties of zerumbone. Our findings conclude that zerumbone from dietary ginger can be considered to develop the food-based anticancer drugs for treating the non-small cell lung cancer. Because ginger ingredients have been widely consumed as functional foods for centuries, our study emphasized the vital relationship between foods and health, and inclusion of zerumbone in preparation of new functional foods.
Conflict of interest statement
The authors have no conflicts of interest to declare.
This work was supported by the grants, MOST-103-2320-B-039-038-MY3, NSC-101-2320-B-039-050-MY3, NSC-103-2622-B-039-001-CC2, 102-ASIA-17 and CMU 102-ASIA-22 from the Ministry of Science and Technology (MOST), National Science Council (NSC), Asia University and China Medical University (CMU), Taiwan. We thank Dr. Ju-Hong Jeon kindly giving Smads (3TP-Luc reporter vector) plasmid.
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Figure legends
Fig. 1 Effect of zerumbone and/or TGF-β1 on cell viability of human lung cancer A549 cells. (A) Chemical structure of zerumbone. Cells were treated with (B) TGF-β1 (1-20 ng/mL) for 24 h or (C) zerumbone (10-80 μM) for 2 h. (D) Cells were pretreated with zerumbone (10 or 20 μM for 2 h), and then stimulated with or without TGF-β1 (2 ng/mL) for 24 h. The effects of zerumbone alone and along with TGF-β1 were determined by MTT assay. The cell viability (%) was calculated as follows: (A570 of treated cells/A570 of untreated cells) × 100. The results are presented as the mean SD of three assays. Significant at *p < 0.05; **p < 0.01 compared to control cells; significant at #p < 0.05 compared to TGF-β1-treated cells.
Fig. 2 Zerumbone blocks TGF-β1-induced cell scattering and EMT through up-regulation of epithelial marker, E-cadherin in A549 cells. Cells were pretreated with zerumbone (10 or 20 μM) for 2 h, and then stimulated with or without TGF-β1 (2 ng/mL) for 24 h. (A) Morphological changes in A549 cells were examined by phase-contrast microscope (200× magnification). (B) Epithelial (E-cadherin) marker proteins were monitored by Western blot. Relative changes in protein bands were measured using densitometric analysis with the control being 100 %. (C) Transcriptional activity of E-cadherin was monitored by luciferase reporter assay. The results are presented as the mean SD of three assays. Significant at *p < 0.05; **p < 0.01compared to control cells; significant at #p < 0.05; ##p < 0.01 compared to TGF-β1-treated cells.
Fig. 3 Immunofluorescence analysis for E-cadherin protein in zerumbone pretreated TGF-β1-stimulated A549 cells. Cells treated with zerumbone (10 or 20 μM) for 2 h prior to TGF-β1 (2 ng/mL) stimulation for 24 h. The presented photomicrographs and histograms are from one of the representative experiment repeated three times with similar results. Significant at *p < 0.01 compared to control cells; significant at #p < 0.05; ##p < 0.01 compared to TGF-β1-treated cells.
Fig. 4 Zerumbone attenuates TGF-β1-activated EMT through the inhibition of Smad2 signaling pathways in A549 cells. Cells were pretreated with zerumbone (20 μM) for 2 h, and then stimulated with or without TGF-β1 (2 ng/mL) for 0.25-24 h. (A) Zerumbone-induced modulation of Smad2 and Smad3 proteins were determined by Western blot. The phosphorylated Smad2 (p-Smad2) and Smad3 (p-Smad3) levels were evaluated using specific phosphorylation antibodies. Relative changes in protein bands were measured using densitometric analysis with the control being 1.0-fold. (B) Transcriptional activity of Smads (3TP-Luc) was monitored by luciferase reporter assay. The histograms shown are from one of the representative experiment repeated three times with similar results. The results expressed as the mean ± S.D of three independent assays. Significant at *p < 0.01 compared to control cells; significant at #p < 0.05; ##p < 0.01 compared to TGF-β1-treated cells
Fig. 5 Zerumbone inhibits TGF-β1-induced cell migration in A549 cells. (A) Cells were pretreated with zerumbone (10 or 20 M) or vehicle control (0.1% DMSO) for 2 h and washed with PBS. Then migration of cells with or without TGF-β1 (2 ng/mL) stimulation after 24 or 48 h treatment was detected. Cells that migrated to the lower membrane were photographed (200 × magnification). (B) The percentage of migrated cells was quantified and expressed relative to untreated cells (control), which represented 100%. To quantify migration, cells were counted in three microscopic fields per sample. The results are presented as the mean SD of three assays. Significant at *p < 0.01 compared to control cells; significant at #p < 0.01 compared to TGF-β1-treated cells.
Fig. 6 Zerumbone inhibits TGF-β1-induced invasion in A549 cells. (A) Cells were pretreated with zerumbone (10 or 20 M) or vehicle control (0.1% DMSO) for 2 h and then invasion of cells after were determined after 24 h treatment with or without TGF-β1 (2 ng/mL). Cells invading under the membrane were photographed (200 × magnification). (B) The inhibition of invading cells were quantified and expressed on the basis of untreated cells (control) that represented 1-fold. The results are presented as the mean SD of three assays. Significant at *p < 0.05 compared to control cells; significant at #p < 0.05; ##p < 0.01 compared to TGF-β1-treated cells.
Fig. 7 Effect of zerumbone on colony formation (anchorage-independent growth) in A549 cells. (A) Cells were treated with zerumbone (10 or 20 μM) for 2 h prior to TGF-β1 (2 ng/mL) stimulation for 24 h, and then incubated for 7 days at 37 °C. The ability to proliferate and colonies formation of cells were assayed in soft agar. Colonies were subsequently stained with p-iodonitrotetrazolium violet (1 mg/mL). (B) The percentage of colony formation was calculated by quantitation of numbers of colonies in the absence of zerumbone as 100%. The results are presented as the mean SD of three assays. Significant at *p < 0.01 compared to control cells; significant at #p < 0.05; ##p < 0.01 compared to TGF-β1-treated cells.
Fig. 8 (A) Effect of different concentrations of zerumbone with different incubation periods on cell viability of A549 cells. Cells were treated with or without zerumbone (0-50 μM) for 24-72 h. The effect of zerumbone on cell viability was determined by MTT assay. * Indicates significant difference (p < 0.01) compared to control cells (without zerumbone) at respective time-points. (B-C) Zerumbone mediated autophagy- or apoptosis-regulatory proteins in A549 cells. Cells were treated with or without zerumbone (10-30 μM) for 48 h. The conversion of I to LC3-II, expression of p62 (B), and protein levels of caspase-3 and PARP (C), were determined by
western blot. Protein (50 g) from each sample was resolved by 8–15% SDS-PAGE with β-actin as a loading control. The photomicrographs are from one representative experiment repeated twice with similar results. Relative changes in protein bands were measured using densitometric analysis with the control being 1.0-fold as presented just below the protein bands. The results are presented as the mean SD of three assays.
