Background
Metastatic potential of lung cancer cells has been accepted to be an important cause of high rate of death worldwide [
1]. Knowledge indicates that the process of cancer cell transition from epithelial to mesenchymal phenotypes or epithelial to mesenchymal transition (EMT) plays a dominate role in facilitating metastasis and progression in many types of cancer [
2‐
4]. EMT-phenotypic cancer cells elicit highly metastatic potentials, such as aggressive migratory, invasive and increased tumorigenicity [
2‐
5]. During EMT, epithelial cells undergo remarkable morphological conversion from stone-like epithelial morphology to elongated-like mesenchymal morphology and the crucial hallmarks of EMT are the loss of E-cadherin, a cellular junction protein typically expressed in epithelial cells, and the increase of mesenchymal markers (e.g. N-cadherin, vimentin, snail and slug) [
2‐
5]. Zinc is a trace element implicated in many important cellular processes including structural, functional and signaling of the cells [
6,
7]. Zinc is detected in plasma at the concentrations ranging from 10 to 18 µM [
6,
7] and the concentration of zinc in plasma or tissues is found to be elevated in pathological condition of cancer [
8‐
11]. Importantly, evidence indicates that several zinc influx transporters such as Zrt/Irt-like protein (ZIP) 6 [
12], ZIP7 [
13] and ZIP10 [
14] were shown to be up-regulated in cancer cells and their high levels correlate with aggressive behaviors and poor prognosis. Such data has suggested the roles of zinc in regulation of cancer cell biology. However, effects of zinc on the EMT process of cancer cells are largely unknown.
Recently, the involvement of ROS in EMT process has been continuously revealed [
15‐
17]. Together with the fact that zinc has been shown to regulate cellular redox status of the cells [
18‐
20], it is possible that zinc may affect the cancer cell behaviors via ROS-dependent mechanism. Indeed, the exogenous zinc has a capability to induce ROS production via NADPH oxidase and mitochondria-dependent mechanism [
18‐
20]. Zinc exposure was found to induce translocation of NADPH oxidase subunits to plasma membrane, which is the signature event for NADPH oxidase activation and such an event was inhibited by the addition of NADPH oxidase inhibitor [
7,
19,
20]. Together, we hypothesize that zinc may affect the process of EMT in lung cancer cells. Also, we attempt to clarify the mechanisms involved in zinc-induced EMT. The findings from this study could help fulfill the understanding in tumor cell biology and could provide important information useful for zinc management in cancer patients.
Methods
Cells and reagents
Human lung cancer epithelial H460 cell was obtained from the American Type Culture Collection (ATCC, Manassas, VA). H460 cell was cultured in RPMI 1640 medium in a 5 % CO2 environment at 37 °C. The media was supplemented with 2 mM l-glutamine, 10 % fetal bovine serum and 100 units/ml of penicillin/streptomycin (Gibco, Gaithersburg, MA, USA). Zinc sulfate, dimethyl sulfoxide (DMSO), 2,7-dichlorofluorescein diacetate (DCFH2-DA), dihydroethidium (DHE), hydroxyphenyl fluorescein (HPF), DMNQ (2,3-dimethoxy-1,4-naphthoquinone),3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33342 were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). Mn (ΙΙΙ) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) was obtained from Calbiochem (San Diego, CA, USA). Antibodies for N-cadherin, E-cadherin, vimentin, snail, slug, phosphorylated FAK (Y397), FAK, and β-actin and peroxidase-labeled secondary antibodies were obtained from Cell Signaling Technology, Inc. (Denver, MA). Mouse monoclonal antibodies for active Rho-GTP and Rac1-GTP were obtained from NewEast Biosciences (Malvern, PA, USA). Immobilon Western chemiluminescent HRP substrate was obtained from Millipore, Corp (Billerica, MA, USA) and Thermo Fisher Scientific Inc. (Rockfort, IL, USA).
Cytotoxicity assay
Cell viability was determined by MTT colorimetric assay. Briefly, cells in 96-well plate were incubated with 500 μg/ml of MTT for 4 h at 37 °C. The supernatant was then removed and dimethylsulfoxide (DMSO) was added to dissolve the formazan product. The intensity was spectrophotometrically measured at 570 nm using an ELISA reader (Anthros, Durham, NC, USA). All analyses were performed in at least three independent replicate cultures. The optical density ratio of treated to non-treated control cells was calculated and presented in terms of relative cell viability.
Apoptosis assay
Apoptotic cell death was detected by Hoechst 33342 staining. After specific treatments, cells were stained with 10 µM of the Hoechst 33342 for 30 min at 37 °C. The apoptotic cells having condensed chromatin and/or fragmented nuclei stained by Hoechst 33342 were visualized and scored under a fluorescence microscope (Olympus IX51 with DP70).
Cell morphology characterization
Cell morphology was investigated by seeding the cells at a density of 5 × 104 cells/well onto a 12-well plate for 48 h. The cells were treated with various concentrations of zinc sulfate for 24 h. The cells were then washed with PBS, fixed with 4 % paraformaldehyde in PBS for 10 min at 37 °C, rinsed three times with PBS, and mounted with 50 % glycerol. Cell morphology was then assessed by a phase contrast microscope (Eclipse Ti-U, Nikon, Tokyo, Japan).
Immunofluorescence
Cells were seeded at a density of 1 × 105 cells/well onto coverslips in six-well plate and incubated overnight. After the treatment, the cells on coverslips were fixed with 4 % paraformaldehyde for 30 min and permeabilized with 0.1 % Triton-X for 20 min. Thereafter, the cells were incubated with 3 % bovine serum albumin (BSA) for 30 min to prevent nonspecific binding. The cells were washed and incubated with rabbit anti-Vimentin antibody for 1 h at room temperature. Primary antibody was removed and the cells were washed and subsequently incubated with Alexa Fluor 488 (Invitrogen) conjugated goat anti-rabbit IgG (H + L) secondary antibody for 1 h at room temperature. Samples were washed with PBS then visualized and imaged by fluorescence microscope (Olympus IX 51 with DP70, Olympus America Inc., Center valley, PA).
Western blot analysis
After specific treatments, cells were incubated in lysis buffer containing 20 mM Tris–HCl (pH 7.5), 1 % Triton X-100, 150 mM sodium chloride, 10 % glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 100 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Roche Molecular Biochemicals) for 90 min on ice. The cell lysates were collected, and the protein content was determined using the BCA protein assay kit (Thermo scientific, IL, USA). Equal amounts of proteins from each sample (60 μg) were denatured by heating at 95 °C for 5 min with Laemmli loading buffer and subsequently loaded onto a 10 % SDS-PAGE. After separation, proteins were transferred onto 0.45 μM nitrocellulose membranes (Bio-Rad, Hercules, CA). The transferred membranes were blocked for 1 h in 5 % nonfat dry milk in TBST (25 mM Tris–HCl pH 7.5, 125 mM NaCl, and 0.05 % Tween 20) and incubated with the appropriate primary antibodies at 4 °C overnight. Then, the membranes were washed twice with TBST for 10 min and incubated with horseradish peroxidase-labeled isotype-specific secondary antibodies for 2 h at room temperature. The immune complexes were detected by enhancement with chemiluminescence substrate (Supersignal West Pico; Pierce, Rockfore, IL) and quantified the level of proteins using imageJ software.
Migration assay
Migration was determined by wound healing and transwell assays. For the wound healing assay, a monolayer of cells was cultured in a 96-well plate, and a wound space was made with a 1-mm-wide tip. After rinsing with PBS, the cell monolayers were incubated with the indicated treatments and allowed to migrate for 24 h. Micrographs were taken under a phase contrast microscope (Olympus DP70, Melville, NY), and the wound spaces were measured using Olympus DP controller software. Quantitative analysis of cell migration was performed using an average wound space from those random fields of view, and the percentage of change in the wound space was calculated using the following formula: % change = (average space at time 0 h) − (average space at time 24 h)/(average space at time 0 h) × 100. Relative cell migration was calculated by dividing the percentage change in the wound space of treated cells by that of the control cells in each experiment. For the transwell assay, the cells were seeded at a density of 5 × 104 cells/well onto the upper chamber of a transwell (8 µm pore size) in a 24-well plate in serum-free medium and incubated with various concentrations of zinc. RPMI medium containing 10 % FBS was added to the lower chamber. Following the incubation, the non-migrated cells in the upper chamber were removed by cotton-swab wiping, and the cells that migrated to the underside of the membrane were stained with 10 µg/ml of Hoechst 33342 for 10 min and visualized and scored under a fluorescence microscope (Olympus IX51 with DP70).
Invasion assay
An invasion assay was performed using a 24-well transwell unit with polycarbonate (PVDF) filters (8 µm pore size). The membrane was coated with 0.5 % matrigel on the upper surface of the chamber overnight at 37 °C in a humidified incubator. The cells were plated at a density of 2 × 104 cells per well into the upper chamber of the transwell unit in serum-free medium. Medium containing 10 % FBS was added to the lower chamber of the unit. After incubation with specific test agents for 24 h at 37 °C, the medium in the upper chamber was aspirated, and the cells on the upper side of the membrane were removed with a cotton swab. The cells that invaded to the underside of the membrane were stained with 10 µg/ml of Hoechst 33342 for 10 min, visualized and scored under a fluorescence microscope (Olympus IX51 with DP70).
In vitro 3D tumorigenesis assay
In vitro 3D tumorigenesis was performed in a matrigel-coated 96-well plate. A plate was coated with 0.5 % agarose and left for solidification. The cells were suspended in culture medium containing 4 % matrigel and various concentrations of zinc, and plated at a density of 3 × 102 cells/well onto a agarose-coated plate. Medium containing various concentrations of zinc were replaced every 3 days. After 10 days, the cells were visualized and scored by image analyzer under microscope (Olympus IX51 with DP70). Whole area of each well was captured in one picture and the colonies with more than 25 µm of diameter were quantified.
ROS detection
Intracellular ROS were determined by fluorescence microplate reader and by flow cytometry using the ROS-specific probe, superoxide anions, hydrogen peroxide and hydroxyl radicals were determined by DHE, DCFH2-DA and HPF, respectively. For fluorescence microplate reader, cells were seeded overnight in 96-well plate. Before zinc treatment cells were incubated with 10 μM of dihydroethidium (DHE), dichlorofluorescein diacetate (DCFH2-DA) or hydroxyphenyl fluorescein (HPF) for 30 min at 4 °C, after which they were washed and treated with various concentrations of zinc (0–50 µM) for 1 and 3 h. After incubation, the fluorescence intensity was immediately analyzed by fluorescence microplate reader (SpectraMax M5, Molecular Devices Corp., Sunnyvale, CA, USA) using a 488-nm excitation beam and a 610-nm band-pass filter for DHE, using a 480-nm excitation beam and a 530-nm band-pass filter for detecting DCF fluorescence or using a 490-nm excitation beam and a 515-nm band-pass filter for HPF. For flow cytometry, cells were seeded overnight in six-well plate. Before zinc treatment cells were incubated with 10 μM of DHE, DCFH2-DA or HPF for 30 min at 4 °C, after which they were washed and treated with 50 µM of zinc for 1 and 3 h. After incubation, cells were washed, re-suspended in phosphate-buffered saline (PBS), and immediately analyzed for fluorescence intensity by FACScan flow cytometer (Beckton Dickinson, Rutheford, NJ) using a 488-nm excitation beam and a 610-nm band-pass filter for DHE, using a 480-nm excitation beam and a 530-nm band-pass filter for detecting DCF fluorescence or using a 490-nm excitation beam and a 515-nm band-pass filter for HPF. Mean fluorescence intensity was quantified by CellQuest software (Becton–Dickinson) analysis of the recorded histograms. Relative fluorescence was calculated as a ratio of the treated to the non-treated control fluorescence intensity.
Statistical analysis
All treatment data were normalized to non-treated controls. Data are expressed as the mean ± SD from three or more independent experiments. Multiple comparisons were examined for significant differences of multiple groups, using analysis of variance (ANOVA), followed by individual comparisons with post hoc test. Statistical significance was set at p < 0.05.
Discussion
Accumulating data have guided for a long time that zinc, an essential element composition of numerous proteins [
6‐
8,
29], may play important parts on the basis of cancer cell biology. The plasma zinc level was found to be significantly elevated in certain cancer tissues [
8‐
11]. Zinc-containing compounds seem to be associated with carcinogenesis in lung cancer other cancers [
30,
31]. In detail, zinc chromate was found to increase cytotoxicity, chromosome damage and DNA double strand breaks in human lung epithelial cells, suggesting the roles of zinc-containing compounds in cell toxicity and carcinogenesis [
30]. Besides, the increase of zinc influx transporters such as ZIP6 [
12], ZIP7 [
13] and ZIP10 [
14] has been linked to the aggressive behaviors and poor prognosis of breast cancer [
12‐
14,
32].
Until recently, the knowledge of zinc on the molecular mechanisms of cancer metastasis is still not fully understood especially those regulating EMT. EMT is considered a critical augmenting process of cancer metastasis as it facilitates cancer dissemination in many ways [
2‐
4]. In the process of EMT, cellular phenotypes are altered together with the distinguished expressions of protein markers being changed from epithelial toward mesenchymal types [
2‐
5]. The process facilitates the loss of cell adhesion, increases motility, and survival in detached condition [
2‐
5]. During EMT, an elongated fibroblast-like morphology of the cells is frequently observed. However, indicators like the switching between E-cadherin and N-cadherin, as well as EMT transcription factors snail and slug are more acceptable as hallmarks of EMT [
2‐
5]. In particular, lung cancer H460 cells were shown to undergo EMT in response to various stimuli [
33,
34]. EMT features of H460 cells were characterized by (i) the change of cell morphology from epithelial to fibroblast-liked mesenchymal shape, (ii) the increased EMT markers N-cadherin, vimentin, snail, and slug, together with the reduction of epithelial marker E-cadherin, and (iii) EMT behaviors, including increased migration, invasion and tumorigenic potential [
33‐
38]. In consistent with such studies, our results showed that zinc-treated lung cancer cells displayed the elongated mesenchymal-like shape with the significant increase of EMT markers namely N-cadherin, vimentin, snail and slug (Fig.
2). Also, we found that the E-cadherin was dramatically reduced in response to zinc treatment.
Previous studies indicated that EMT facilitates the cell motility by decreasing cell–cell interaction via E-cadherin, while increases in N-cadherin-mediated steady-state of active Rac1 [
21,
22]. Similar to N-cadherin, Vimentin was shown to increase FAK and Rac1 activities [
23,
24]. We found that zinc could induce EMT, resulting in the increase of cancer cell migration and invasion. The proteins regulating cell motility like FAK, RhoA and Rac1 were found to be activated in response to such up-stream signals (Fig.
3). Also, the EMT event was shown to be a key factor that enhances ability of cancer cells to metastasis by increasing the survival after cell detachment and ability to form tumors [
34,
39]. It has been previously reported that the ability of cancer cells in forming new tumor can be enhanced by EMT as a result from snail augmentation [
25‐
27]. We have supported this fact by the demonstration that treatment of the cells with zinc induced EMT with significant increase of snail increased number and size of tumor colonies in 3D culturing anchorage-independent condition (Fig.
4).
In the previous work, we have reported that the widely used chemical agent triclosan could be able to enhance EMT and aggressive behaviors in anoikis-resistant lung cancer cells [
34]. However, role of endogenous element zinc on such effects has not been clarified. This current study has reported for the first time that zinc significantly induced EMT and tumorigenic potential in lung cancer cells through the enhancement of cellular superoxide anion level. Superoxide anion has been implicated in various biological and pathological processes [
40‐
42]. Evidence has shown that the level of superoxide anion is frequently upregulated in cancer cells and regulates cancer cell proliferation, migration and metastasis [
42‐
46]. Interestingly, we have first revealed the role of such a specific ROS in regulation of EMT in cancer cells, as the EMT could be induced by addition of superoxide anion generator (Figs.
6,
7). The EMT mediated by zinc treatment was abolished by the superoxide anion inhibitor. These findings not only provide the evidence of endogenous element in regulation of cancer biology, but also add the fact involving specific ROS roles on EMT process of cancer cells. The involvement of ROS signaling on cancer metastasis and EMT has garnered increasing attentions [
15‐
17,
47]. The EMT-related transcription factor snail was shown to be sensitive to the balance of cellular ROS status [
47,
48].
Interestingly, zinc was previously addressed to have the potential to interfere with redox status of the cells by inducing oxidative stress [
18‐
20]. In neurons, intracellular zinc is shown to trigger the ROS production during the process of neuron damage [
49,
50]. Besides, the elevation of intracellular zinc was shown to induce superoxide anion production from the function of 12-lipoxygenase (12-LOX) enzyme [
49,
50]. Interestingly, zinc was demonstrated to promote Hep-2 cancer cell apoptosis by stimulating oxidative stress [
18]. Our results showed that zinc could increase superoxide anion in the human lung cancer cells (Fig.
5). The superoxide anion in such cases was suggested to be generated through NADPH oxidase system [
19,
20]. Although studies have indicated that hydrogen peroxide can also mediate EMT in human malignant mesothelioma and human ovarian cancer cells [
51,
52], treatment of the zinc in our system caused no effect on the cellular level of hydrogen peroxide (Fig.
5). We further investigated the role of superoxide anion on EMT using the superoxide anion inducer DMNQ. DMNQ is known to induce superoxide anion generation via NADPH oxidase activity [
53,
54]. We found that treatment of the cells with DMNQ significantly increased the protein hallmarks of EMT as well as metastatic potentials (Figs.
6,
7). Such roles of superoxide anion on EMT were linked with the results of zinc, suggesting that zinc mediates the EMT phenotypes via the production of cellular superoxide anion. These results were confirmed by the ROS inhibitory experiment. Addition of MnTBAP in the zinc-treated cells was shown to abolish superoxide anion induction as well as EMT phenotypes in response to zinc treatment (Figs.
6,
7), strongly indicating that the effect of zinc on EMT was regulated via superoxide-dependent mechanism.