Background
Magnolin is the major component abundantly found in the dried buds of the magnolia flower, Shin-Yi, which has been traditionally used as an oriental medicine to treat nasal congestion associated with headaches, sinusitis, inflammation, and allergic rhinitis [
1]. A previous study has indicated that topical application of the
Magnolia flos (flosculous: a small budding flower) extract inhibits passive cutaneous anaphylaxis induced by anti-dinitrophenyl (DNP) IgE in rats [
2]. Recent studies have demonstrated that magnolin inhibits the production of tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) by inhibiting extracellular signal-regulated kinases (ERKs) [
3,
4], which are key signaling molecules in the regulation of cell proliferation, transformation [
5] and cancer cell metastasis [
6]. Our previous results have demonstrated that magnolin targeting ERK1 (IC
50 87 nM) and ERK2 (IC
50 16.5 nM) inhibits cell transformation induced by tumor promoters such as epidermal growth factor (EGF) [
5]. To date, no direct evidence regarding the inhibitory effects of magnolin on metastasis has been provided.
The 90 kDa ribosomal S6 kinases (p90RSKs: RSKs) are a family of serine/threonine kinases activated by the Ras/MEKs/ERKs signaling pathway, which responds to diverse extracellular stimuli [
7]. RSK2 is a member of the RSK family and is phosphorylated at the C-terminal kinase and linker domains by ERK1/2 [
8] and at the N-terminal kinase domain by phosphoinositide-dependent kinase 1 (PDK1) [
9]. Activated RSK2 transduces its activation signal to various downstream target proteins including transcription and epigenetic factors [
10‐
12], kinases [
13], and scaffolding proteins such as nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α (IκBα) [
14], and regulates diverse cellular activities involved in cell proliferation, transformation and motility [
15]. For instance, our previous results have demonstrated that the enhanced cAMP-dependent transcription factor 1 (ATF1) activity, caused by the epidermal growth factor (EGF)-mediated Ras/ERKs/RSK2 signaling pathway, induces cell proliferation and transformation [
16]. The increased NF-κB transactivation activity, resulting from the RSK2-IκBα signaling pathway, modulates cell survival induced by the FAS-mediated death signaling pathway [
13]. A recent report demonstrates that RSK2 promotes the invasion and metastasis of head and neck squamous cell carcinoma cells in humans [
17]. Therefore, the Ras/ERKs/RSK2 signaling axis may be a key signaling pathway in the regulation of cell proliferation and transformation, and in cancer cell metastasis.
Nuclear factor-κB (NF-κB) is a ubiquitous nuclear transcription factor composed of p65 (Rel A), p68 (Rel B), p75 (c-Rel), p50 and p52 [
18]. In the absence of cellular stimulation, NF-κB is located in the cytoplasm and forms a complex with specific inhibitors of NF-κB (IκBs). Upon cell stimulation by growth factors and proinflammatory cytokines, IκBα is phosphorylated by IκBα kinase (IKK), leading to ubiquitination and degradation [
19]. Following degradation of IκBα, NF-κB translocates to the nucleus and effects the expression of genes involved in cell proliferation, invasion and metastasis [
19]. Recently, we identified an alternative signaling pathway regulating NF-κB activation, in which RSK2 phosphorylates IκBα at Ser32, promoting the ubiquitination-mediated degradation of IκBα [
20]. Due to the fact that ERK1 and 2 are direct upstream kinases of RSK2 [
8], targeting ERK1/2 with small molecules may be the focus in the development of a drug acting as a metastatic inhibitor.
The mitogen-activated protein kinase (MAPK) family is comprised of three subfamilies including ERKs, p38 kinases and c-Jun N-terminal kinases (JNKs), which play a key role in the regulation of cellular responsiveness by the diverse extracellular stimuli such as growth factors, peptide hormones, and environmental stressors such as ultraviolet light [
13,
21,
22]. The ERKs/RSK2 signaling axis plays a pivotal role in cell proliferation, differentiation, survival, and transformation [
8,
10,
13,
15,
21], in addition to cell migration through the induction of matrix metalloproteinases (MMPs), which are the enzymes that degrade the extracellular matrix, such as collagen and gelatin, to facilitate the metastasis of cancer cells [
6]. Recently, our research group found that magnolin, a major component of
Magnolia flos (Shin-Yi) that has been traditionally used as an oriental medicine to treat headaches, nasal congestion and inflammatory reactions [
23], inhibits the Ras/ERKs/RSK2 signaling axis by targeting the active pocket of ERK1 and ERK2 with IC
50 values of 87 nM and 16.5 nM, respectively [
5]. Furthermore, we found that AP-1 and NF-κB transactivation activities were downregulated by the inhibition of ERK1/2-mediated RSK2 activity [
5,
20], suggesting that magnolin may suppress the gene expression of
Cox-2, an enzyme that plays an important role in cancer cell proliferation, motility and metastasis [
24]. Generally, metastasis is complicated multiple biological processes including 1) loss of adhesion involved during epithelial to mesenchymal transition (EMT), 2) increased motility and invasiveness to achieve intravasation, 3) circulation through blood vessels and lymph nodes, and 4) attachment to blood vessels followed by extravasation [
25]. Eventually, the metastatic cancer cells succeed in colonizing on distant organ tissues, which causes more than 90 % of cancer deaths [
26]. However, the molecular mechanisms behind magnolin-mediated cell migration and invasion are not yet clearly understood.
Methods
Reagents and antibodies
Chemical reagents such as NaCl, Tris, sodium dodecyl sulfate (SDS) and buffer preparations were purchased from Sigma-Aldrich chemical Co. (St. Louis, MO, USA). Recombinant EGF was purchased from BD Sciences (San Jose, CA, USA). Antibodies against phospho-IκBα, total-IκBα, N-cadherin, β-actin, MMP-2, MMP-9, E-cadherin, COX-2, and total-RSK2 were purchased from Cell Signaling Technology (Beverly, MA, USA), Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Thermo Fisher Scientific Inc. (Waltham, MA, USA). Cell culture media and other supplements were purchased from Life Science Technology (Rockville, MD, USA) and Corning (Corning, NY, USA).
Magnolin
Magnolin was extracted from the dried flower buds of
Magnolia fargesii in accordance with the method established by Lee
et al., (Korea Patent # 10-0321212-0000) [
27] and confirmed a purity of >99.0 % using high-performance liquid chromatography (HPLC), which was generously provided by Dr. SR Oh of the Korean Research Institute of Bioscience and Biotechnology (KRIBB). Magnolin was prepared as a stock solution (100 mM: 1000X) by dissolving in DMSO obtained from Sigma-Aldrich Co. LLC., (St. Louis, MO, USA), after which it was aliquoted and stored at −20 °C. The magnolin was freshly diluted in DMSO before utilization, and the cells were treated upon medium exchange with magnolin premixed cell culture medium, in which the DMSO concentration did not exceed 0.1 % of the total volume.
Cell culture and transfection
JB6 Cl41 cells purchased from ATCC were cultured with 5 % FBS-MEM, and RSK2+/+ and RSK2−/− mouse embryonic fibroblasts (MEFs) were cultured with 10 % FBS-DMEM, containing penicillin/streptomycin, at 37 °C in a 5 % CO2 incubator. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee at the Catholic University of Korea (approval number: 2014–0046). A549 and NCI-H1975 human lung cancer cells, purchased from ATCC, were cultured with 10 % FBS-F12K and 10 % FBS-RPMI 1640, respectively, according to the guidelines of Institutional Laboratory Safety. The cells were maintained by passage at 80-90 % confluence, and the media was changed every other day. Transfection of the various expression vectors was carried out using jetPEI (Polyplus-Transfection Inc., New York, NY, USA) according to the manufacturer’s protocol.
Cell migration and invasion assay
JB6 Cl41 (7 × 104), A549 (7 × 104) and NCI-H1975 (7 × 104) cells, and RSK2+/+ (7 × 104) and RSK2−/− (7 × 104) MEFs were seeded into culture-inserts (ibidi GmbH, Martinsried, Germany) and cultured overnight. The cells were treated with mitomycin-C (10 μg/ml) for 2 h, and the culture-inserts were removed to offer a cell-free gap. The cells were treated with the indicated doses of magnolin either in the presence or absence of EGF for 12 or 24 h, and cell migration was observed under a light microscope. The migrated area was measured using the Image J computer software program (v. 1.45). To measure the magnolin effect on cancer cell invasion, a matrigel-coated invasion chamber (Corning Incorporated, Coring, NY, USA) was used. Briefly, A549 or NCI-H1975 (2.5 × 104) cells were seeded into an insert chamber with FBS-free media supplemented with the indicated doses of magnolin, and cultured in 24-well plates supplemented with complete media for the appropriate time period. The cells were fixed with 4 % formaldehyde, permeabilized with methanol and stained with crystal violet. The stained cells were observed under a light microscope and those that had migrated were counted.
Gelatin zymography
MMP-2 and −9 activities were evaluated by gelatin zymography using the cell culture supernatants. Briefly, A549 cells (4 × 105) were seeded into 60 mm dishes, cultured and treated with the indicated doses of magnolin for 24 h. The culture supernatants were harvested, and 20 μg of protein from each sample were loaded on a polyacrylamide gel containing 0.2 % gelatin. The gel was washed with 2.5 % Triton X-100 buffer for 20 min, and then incubated for 24 h at 37 °C in renaturing buffer [50 mM Tris-Cl (pH 7.5), 10 mM CaCl2, 1 μM ZnCl2, 0.01 % NaN3]. The gels were stained with Coomassie Brilliant Blue and destained in methanol/acetic acid.
Immunocytofluorescence (ICF)
A549 cells (6 × 104) were seeded into 4-chamber slides, cultured and treated with the indicated doses of magnolin for 24 h. The cells were fixed with 4 % formalin, blocked in 1 % BSA/Tween-20/1X PBS at room temperature for 1 h, and hybridized with anti-N-cadherin primary and Flamma Fluors 552- or Alexa-488-conjugated secondary antibodies (BioActs, Incheon, Gyeonggi-do, Korea). The slides were mounted with FluoroshieldTM-DAPI (Sigma-Aldrich, St. Louis, MO, USA). The N- and E-cadherin protein levels were visualized under a LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
Real-time PCR (RT-PCR)
A549 cells (5 × 105) were seeded into 60 mm dishes, cultured overnight and treated with the indicated doses of magnolin for 24 h. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and quantitative gene expression levels of MMP-2 and −9 were measured by real-time polymerase chain reaction (PCR) using a specific primer set, MMP-2 (Hs01548727_m1) and MMP-9 (Hs00234579_m1), a GAPDH specific real-time primer set (4352934E), and a TaqMan RNA-to-CT 1-step kit (applied Biosystems, Foster City, CA, USA) according to the manufacturer’s recommended protocol. The CT values of MMP-2 and MMP-9 RNA expression were normalized to the CT values of GAPDH as an internal control to ensure equal RNA utilization.
Reporter gene assay
JB6 Cl41 (2 × 104) cells stably expressing an NF-κB- or Cox-2-promoter luciferase reporter plasmid, and A549 cells (2 × 104) cells stably expressing an MMP-2 or an MMP-9 promoter luciferase reporter plasmid were seeded into a 24-well plate and cultured overnight. The cells were starved for 16 h, pretreated with the indicated doses of magnolin for 30 min, and then co-treated with EGF (10 ng/ml) at the indicated doses of magnolin for 24 h. The cells were disrupted, and the firefly luciferase activities were measured using a VIXTOR X3 fluoro/luminometer (Perkin Elmer Inc., Waltham, MA, USA).
Western blotting
Samples containing equal amounts of proteins as indicated were resolved by SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5 % skimmed milk/1X PBS/0.5 % Tween 20 at room temperature for 1 h, and hybridized with the specific primary and HRP-conjugated secondary antibodies as indicated. The proteins were visualized by an enhanced chemiluminescence (ECL) detection system (Amersham Bioscience Corp., Piscataway, NJ, USA).
Discussion
Oriental medicinal herbs contain many useful compounds, and have been widely used to identify novel compounds that may have therapeutic value in the treatment of human diseases. For instance, myricetin and quercetin from dietary herbs and epigallocatechin gallate from green tea inhibit cell proliferation and transformation [
36], highlighting the importance of efforts to identify natural compounds that inhibit the ERKs/RSKs signaling pathway, while suppressing the MAPK pathway in a non-toxic manner [
37]. Early buds of the magnolia flower are an oriental medicinal herb and traditionally used to treat inflammation-mediated human diseases including empyema, nasal congestion, sinusitis and allergic rhinitis [
1]. Recent reports have provided evidence that magnolin inhibits the expression of cell adhesion molecules including intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 [
38]. However, although magnolin has shown diverse effects on human diseases, the molecular targets of magnolin had not yet been identified. Recently, our research group found that ERK1 and ERK2 are the molecular targets of magnolin, which inhibited their kinase activity with IC
50 values of 87 nM and 16.5 nM, respectively, by competing with ATP in an active pocket [
5]. Furthermore, our previous results have demonstrated that ERK1/2-mediated RSK2 activation modulates NF-κB activity by phosphorylation of IκBα at Ser32 [
20]. These results provide us with the rationale to consider that magnolin may show effectiveness on cell migration and cancer metastasis. In pancreatic cancer, the NF-κB signaling pathway plays an important role in EMT and metastasis [
39,
40]. Moreover, NF-κB activation induces classical EMT marker changes and the promotion of cell migration and invasion [
41], indicating that ERK/RSK2/NF-κB signaling may play a key role in cell migration and invasion. Our results support the notion that RSK2 activity modulates NF-κB activity (Fig.
1c and
d). Importantly, the knockdown and knockout of RSK2 attenuated cell migration (Fig.
4c and
d), which was similarly observed upon magnolin treatment of A549 and NCI-H1975 lung cancer cells (Fig.
3a and
b). These results demonstrate that ERK inhibition by magnolin suppresses RSK2-mediated NF-κB activity, resulting in suppression of cell migration and invasion in cancer cells.
The cellular program called EMT is accompanied by profound changes in cell characteristics that enable the epithelial cells to detach from tight junctions, change the cell’s shape and polarity, delaminate, and migrate [
42]. A great number of growth factors and signaling pathways have been associated with EMT induction, including EGF through the JAK pathway and the ERK/MAPK signaling pathway [
43,
44]. Previous reports have indicated that coffee or chlorogenic acid abolished CT-26 metastasis to the lung by blocking ERK/AP-1 and ERK/NF-κB signaling pathways [
45]. Our results demonstrate that EGF stimulation induces cell migration in JB6 Cl41 cells (Fig.
2d). The role of RSK2 in EGF-induced cell migration was confirmed using RSK2
+/+ and RSK2
−/− MEFs, that RSK2 deficiency abrogated EGF-induced wound healing (Fig.
4d). In A549 cancer cells, we further confirmed that knockdown of RSK2 using RSK2 sh-RNA suppressed cell migration (Fig.
4c). Interestingly, we found that RSK2 knockdown inhibited MMP-2 and N-cadherin and enhanced E-cadherin (Fig.
4c), however, unexpectedly, there were no significant changes observed in Snail and Vimentin (Fig.
4c). Similar results were observed with Snail, but not with Vimentin, in RSK2
+/+ and RSK2
−/− MEFs (Fig.
4d). The gene expression of Snail is dependent on ERKs activity through the ERK/Fra-1/c-Jun signaling pathway [
46] and the ERK/ELK-1 signaling pathway [
47]. Our previous results have demonstrated that RSK2 deficiency dramatically increased total protein levels and phosphorylation sensitivity of ERK1/2 by EGF treatment [
13]. Thus, we suggest that the no change in Snail is due to the reactivation of ERKs by the activation of the RSK2 feedback loop. Based on this hypothesis, it is possible to explain that magnolin inhibited cell migration and invasion by downregulating ERK-mediated Vimentin protein level by downregulating the RSK2-mediated NF-κB signaling pathway. Taken together, these results demonstrate that RSK2 mediates EGF-induced cell migration signaling through the ERKs/RSK2 signaling pathway.
The wound healing assay is a well-adapted strategy to evaluate cancer cell metastasis
ex vivo [
48]. The RSK2 function in cancer metastasis has been observed from head and neck squamous cell carcinoma (HNSCC) in cancer patients [
17]. This evidence was proved by a xenograft metastasis experiment showing that knockdown of RSK2, but not RSK1, reduced the metastasis of human HNSCC cells [
17]. Furthermore, RSK protein levels are important in determining whether cancer cells have the capability to metastasize. RSK1-silencing enhances
in vitro cell migration, and human patient samples of metastatic lung cancer have lower RSK1 expression levels compared with non-metastatic cancer tissues [
49]. In contrast, cancer tissue analysis from HNSCC showed a positive relationship between the metastatic ability and RSK2 protein levels [
49]. Our previous results have demonstrated that total- and activated-RSK2 protein levels were observed in a human tissue array of skin cancers [
8,
22]. Importantly, our
ex vivo study demonstrated that RSK2 protein levels were more enhanced in skin cancer cells such as malignant melanoma compared with squamous cell carcinoma and premalignant immortalized cells [
22]. The signaling study of RSK2 indicates that RSK2 can phosphorylate GSK3β at Ser9, resulting in enhanced cell survival from stresses such as calcium and UV irradiation [
13]. Due to the fact that RSK2 is phosphorylated by ERK1 and 2, but not by p38 kinase, ERK1 and 2 inhibitors may be useful compounds to inhibit cancer cell metastasis. Magnolin is a potent natural compound, having strong inhibitory effects on ERK1 and 2 by competing with ATP in the active pockets [
5], and we believe that magnolin has a potential application in human cancer prevention and metastasis.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CJL performed the experiments; SMY and KC conducted real-time RT-PCR and Western blotting, JHS and JHJ performed cell migration assays; MHL and YJS were involved in the design of the study; HSL and ARO provided the magnolin and were involved in data analysis; YYC managed the project; all authors read and approved the final manuscript.