Article pubs.acs.org/jnp
Zerumbone Suppresses Osteopontin-Induced Cell Invasion Through Inhibiting the FAK/AKT/ROCK Pathway in Human Non-Small Cell Lung Cancer A549 Cells Chi Gu Kang, Hyo-Jeong Lee, Sung-Hoon Kim, and Eun-Ok Lee* Cancer Preventive Material Development Research Center, College of Korean Medicine, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of Korea ABSTRACT: Lung cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths in the United States and Korea. We have previously demonstrated that osteopontin (OPN) induces cell invasion through inactivating cofilin. Inactivation of cofilin is mediated by the FAK/AKT/Rho-associated kinase (ROCK) pathway in human nonsmall cell lung cancer (NSCLC) cells. Zerumbone (1) has been shown to exert anticancer activities. In this study, whether and how 1 affects OPN-induced cell invasion was determined in NSCLC A549 cells. Results from Boyden chamber assays suggested that OPN induced invasion of A549 cells and that 1 strongly suppressed this activity without affecting cell viability. Compound 1 effectively inhibited OPN-induced protein expression of ROCK1, the phosphorylation of LIM kinase 1 and 2 (LIMK1/2), and cofilin. In addition, immunofluorescence staining showed that OPN caused a significant increase in lamellipodia formation at the leading edge of cells. However, 1 dramatically decreased OPN-induced lamellipodia formation. Compound 1 impaired OPN-induced phosphorylation of FAK and AKT, as determined by Western blot analysis. Taken together, these results suggest that 1 causes considerable suppression of OPNinduced cell invasion through inhibiting the FAK/AKT/ROCK pathway in NSCLC A549 cells.
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induced invasion of breast and pancreatic cancer cells.13 However, the effect of 1 on the invasion of lung cancer is not yet fully understood. The anti-invasive effect of 1 and its underlying molecular mechanisms in lung cancer have yet to be determined.
ung cancer is the most common form of cancer and is expected to account for 14% and 13% of all new cancers in men and women, respectively, in the United States.1 As the leading cause of cancer deaths, lung cancer is expected to account for 28% and 26% of all cancer deaths in men and women, respectively, in the United States in 2015.1 In Korea, lung cancer is also the leading cause of cancer deaths, accounting for 22.6% of all cancer deaths in 2012.2 Despite a rapid advance in understanding of the mechanisms of lung carcinogenesis, there is still no effective clinical treatment for cancer patients. Thus, novel targets and agents for use in the chemoprevention of lung cancer are sorely needed. Cancer chemoprevention is defined as the use of dietary or pharmaceutical interventions to slow or reverse the progression of premalignancy to invasive cancer.3 Due to their anti-invasive and antimetastatic actions on tumors, some natural products such as curcumin, resveratrol, and (−)-epigallocathechin-3gallate have been assessed as chemopreventive agents for lung cancer.4,5 Zerumbone (1), a natural cyclic sesquiterpene, was first isolated in 1956 from rhizomes of a Southeast Asian ginger, Zingiber zerumbet Smith,6 which has been used traditionally for the treatment of stomachache, toothache, fever, and indigestion.7 Compound 1 exhibits many biological activities such as antiinflammatory, antihyperglycemic, antioxidative, and antiatherosclerotic activities.8−11 A limited number of studies reported that 1 suppresses TNF-induced invasion by the inhibition of NF-κB activation in H1299 lung cancer cells,6 IL1β-induced cell migration and invasion by the inhibition of IL-8 and MMP-3 expression in breast cancer cells,12 and CXCL12© XXXX American Chemical Society and American Society of Pharmacognosy
Osteopontin (OPN) is secreted by numerous human cancers and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of chemokine-like calcified extracellular matrix-associated glyco-phosphosialo protein.14 OPN has been identified as one of the important mediators of stroma−tumor interaction, contributing to tumor growth, metastasis, and angiogenesis.15,16 OPN is well-known to induce oncogenic and metastatic potential by upregulating various signaling pathways mediated by its cell surface receptors, integrins, and CD44 in several cancers,15 Cancer metastasis is a multiple cellular process involving adhesion, migration, invasion, and morphogenesis, which is associated with the dynamics of actin cytoskeleton.17,18 Rho family small GTPases such as Rac, Rho, and Cdc42 play central role in regulation of actin cytoskeleton reorganization. Of these Received: September 14, 2015
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DOI: 10.1021/acs.jnatprod.5b00796 J. Nat. Prod. XXXX, XXX, XXX−XXX
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upregulating stromal cell derived factor-1α/CXC chemokine receptor 4/matrix metalloproteinase (MMP) 2 system.24 In addition, OPN stimulates the motility of breast cancer cells through αvβ3 integrin-mediated phosphoinositide-3-kinase (PI3K)/Akt/nuclear factor κB-25 and c-Src/epidermal growth factor receptor/AP-1-dependent26 secretion of urokinase-type plasminogen activator. Some previous studies reported that 1 exhibits a role in the inhibition of cell invasion in various types of cancer cells including H1299 lung cancer cells.6,12,13 To determine whether 1 inhibits cell invasion induced by OPN in NSCLC A549 cells, cell invasion assays were performed for 24 h after preincubation with 1 for 30 min using a Boyden chamber. Consistent with our previous report,21 1 nM of OPN stimulation for 24 h strikingly induced the invasive behavior of A549 cells into matrigel by 250%, which was greatly suppressed in the presence of 1 at 5 and 10 uM by 48% and 69%, respectively (Figure 2). These results demonstrate that 1 has an anti-invasive effect on OPN-induced A549 cells.
downstream effectors, Rho-associated kinase (ROCK) and p21activated kinases (PKAs) phosphorylate and activate LIM kinases (LIMKs). Activated LIMKs phosphorylate cofilin at Ser3 and thus inhibit its actin depolymerization activity, thereby leading to an actin polymerization.19,20 Rho family small GTPases-indued actin polymerization at the front and leading edge forms sheet-like structures known as lamellipodia, which pull cells forward to induce migration and invasion.19,20 Therefore, the ROCK or PAK/LIMK/cofilin pathway is a crucial event in cancer migration and invasion. We have previously demonstrated the following results.21 (1) OPN inactivates cofilin through the ROCK/LIMK pathway but not the PAKs/LIMK pathway, (2) which is involved in the FAK/AKT pathway. (3) OPN induces lamellipodia formation at the leading edge of cells and cell invasion. (4) Finally, results from inhibitors treatment suggested that OPN induces cell invasion through the ROCK/LIMK/cofilin pathway mediated by the FAK/AKT pathway in human nonsmall cell lung cancer (NSCLC) A549 cells. OPN and OPN-regulated signaling pathways may be potentially important targets for developing novel molecular diagnostics for the effective management of cancer. Here, we studied the ability of 1 to inhibit OPNinduced invasion of NSCLC A549 cells to develop a novel antiinvasive agent.
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RESULTS AND DISCUSSION Effect of Zerumbone (1) on the Viability of A549 Cells. We tested 1 for cytotoxicity because 1 enhances TRAILinduced apoptosis in colon cancer cells,9 causes cell cycle arrest and Bax/Bak-mediated apoptosis in breast cancer cells,22 and inhibits proliferation and induces apoptosis through suppressing signal transducer and activator of transcription 3 activation in renal cell carcinoma.23 To determine whether 1 is cytotoxic to the human nonsmall cell lung cancer (NSCLC) cell line A549, cells were treated with various concentrations of 1 in serum-free media for 24 h, and cell viability was evaluated by MTT assay. The viability of A549 cells significantly decreased in the presence of 1 at 20 μM. However, as shown in Figure 1,
Figure 2. Zerumbone (1) suppresses the osteopontin-induced invasion of A549 cells. The cell invasion assay was performed using a Boyden chamber with a Matrigel-coated filter. Compound 1pretreated cells and 1 nM of OPN were loaded into the upper wells and lower wells, respectively. (A) After incubation at 37 °C for 24 h, the cells were stained and then randomly chosen fields were photographed at 200×. Scale bar = 100 μm. (B) The number of cells invaded to the lower surface was counted. Data are shown as the mean ± SD of two independent experiments by analysis of Student’s t test. *** p < 0.001 vs untreated control and ### p < 0.001 vs OPNtreated control.
Figure 1. Effect of zerumbone (1) on the cell viability of A549 cells. Cells were treated with 1 for 24 h and cell viability was evaluated by MTT assay. Data are shown as the mean ± SD of three independent experiments by analysis of Student’s t test. ** p < 0.01 vs untreated control.
there was no cytotoxic effect of 1 to A549 cells at concentrations as high as 10 μM. To exclude the influence of cell viability, noncytotoxic concentrations (≤10 μM) were used for all sbusequent experiments. Zerumbone (1) Suppresses Osteopontin-Induced Invasion of A549 Cells. OPN plays an important role in all stages of cancer progression including invasion, metastasis, and angiogenesis.15 For example, Zhang et al. have shown that OPN induces the invasion of hepatocellular carcinoma cells by
Zerumbone (1) Inhibits Osteopontin-Induced the ROCK/LIMK/Cofilin Signaling Pathway in A549 Cells. The PAK/LIMK/cofilin signaling pathway contributes to the migration and invasiveness of NSCLC, especially in squamous cell carcinoma.27 However, previous studies have shown that OPN inactivated cofilin by phosphorylation, which was B
DOI: 10.1021/acs.jnatprod.5b00796 J. Nat. Prod. XXXX, XXX, XXX−XXX
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mediated by the ROCK/LIMK pathway but not by the PAK/ LIMK pathway in NSCLC.21 To investigate whether 1 affects this pathway, Western blot analysis was carried out after OPN stimulation in the presence or absence of 1 for 24 h. As expected, OPN increased the expression of ROCK1, and the phosphorylation of LIMK1/2, which in turn increased the phosphorylation of cofilin, which is an inactive form. However, 1 effectively decreased OPN-induced this pathway in a dosedependent manner without affecting their total forms (Figure 3). These results verify that 1 may act through the ROCK/ LIMK/cofilin pathway to suppress the invasion of A549 cells.
Figure 4. Zerumbone (1) decreases osteopontin-induced lamellipodia formation in A549 cells. Cells were seeded in a chamber slide and treated with 1 nM OPN for 24 h in the presence or absence of 1. After they were fixed and permeabilized, the cells were incubated with rhodamine phalloidin (F-actin, red) and counterstained with DAPI (nucleus, blue), and visualized under a confocal microscope at 600×. Scale bar = 40 μm.
apoptosis. Among these pathways, it is well-known that FAK and AKT affect the motility of various types of cancer cells. It has been reported that SRPX2 expression correlates with the FAK signaling, contributing to cell migration and invasion in pancreatic ductal adenocarcinoma,28 and that 3′3-diindolylmethane blocks hepatocellular carcinoma metastasis by inhibiting FAK-mediated MMP2/9 activation.29 Some studies showed that PI3K/AKT-induced MMP2/9 expression and activation causes cell migration and invasion.30,31 OPN, via integrin, activates FAK and PI3K/AKT signaling to promote cell migration and invasion in lung and breast cancer cells.25,32 In addition, it has been demonstrated that FAK/AKT mediates cell invasion by OPN stimulation through regulating the ROCK/LIMK/cofilin signaling and lamellipodia formation in NSCLC A549 cells.21 Here, Western blot analysis was performed to define whether the effect of 1 on OPN-induced invasion of A549 cells resulted from impairing the FAK/AKT pathway. As shown in Figure 5, OPN caused increases in the phosphorylation of FAK and AKT, which were effectively decreased after treatment with 1 without affecting their total forms. These results indicate that the FAK/AKT pathway is associated with inhibiting OPN-induced invasion by 1 treatment. In conclusion, the observations herein reported demonstrate for the first time that 1 can suppress OPN-induced invasive ability of NSCLC A549 cells by reducing lamellipodia formation at noncytotoxic concentrations. This effect is mediated by the FAK/AKT pathway, which in turn inhibits the ROCK/LIMK/cofilin pathway. These results provided new insight into the function of 1 through an anti-invasive mechanism and suggest that 1 is worthy of further study as an agent that may prevent NSCLC metastasis.
Figure 3. Zerumbone (1) impairs osteopontin-induced the ROCK/ LIMK/cofilin signaling pathway in A549 cells. Cells were treated with 1 nM OPN for 24 h in the presence or absence of 1. (A) Equal amount of total proteins (25−30 μg) were analyzed by Western blot using the indicated antibodies. (B) The bands of Western blot were quantified. Data are shown as the mean ± SD of two independent experiments by analysis of Student’s t test. * p < 0.05 vs untreated control and # p < 0.05 vs OPN-treated control.
Zerumbone (1) Reduces Osteopontin-Induced Lamellipodia Formation in A549 Cells. Actin polymerization through actin cytoskeleton reorganization at the leading edge stimulates the formation of sheet-like structures known as lamellipodia, which are crucial for cell migration and invasion toward chemoattractants.19,20 OPN controls actin polymerization by cofilin inactivation,21 which was restored by 1 treatment, as shown in Figure 3. To assess whether 1 affects actin polymerization regulated by cofilin to induce cell invasion, A549 cells were stained with phalloidin for F-actin and counterstained with DAPI to stain nuclei. The results from immunofluorescence assays showed that OPN induced F-actin accumulation and that treatment with 1 dramatically reduced this effect (Figure 4). These results imply that 1 decreases the lamellipodia formation by OPN treatment. Zerumbone (1) Impairs Osteopontin-Induced the FAK/AKT Pathway in A549 Cells. There are numerous and complicated signaling pathways contributing to the cellar process including cell survival, motility, differentiation, and
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EXPERIMENTAL SECTION
General Experimental Procedures. Zerumbone (1) (purity: ≥ 98% as determined by HPLC), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) and the primary antibody for β-actin were purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). Osteopontin (OPN) was obtained from R&D Systems (Minneapolis, Minnesota, U.S.A.). The primary antibodies for ROCK1, LIMK2, phospho-cofilin, cofilin, phospho-FAK and FAK were purchased from Cell Signaling (Beverly, MA, U.S.A.) and phospho-AKT, AKT and C
DOI: 10.1021/acs.jnatprod.5b00796 J. Nat. Prod. XXXX, XXX, XXX−XXX
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cofilin, cofilin, phospho-FAK, FAK, phospho-AKT, AKT, phosphoLIMK1/2, and β-actin overnight at 4 °C, and specific secondary antibodies for 2 h at room temperature. The protein expression was detected by an enhanced chemiluminescence system (GE Healthcare). The bands of Western blot were quantified using ImageJ 1.40g software (National Institutes of Health, Bethesda, MD, U.S.A.). Immunofluorescence Assay. Immunofluorescence for F-actin was assayed as described previously.21,33 Briefly, cells (5 × 104 cells/ well) were seeded in a chamber slide and treated with 1 nM OPN for 24 h in the presence or absence of 10 μM of 1. The cells were then fixed in 4% formaldehyde in PBS (pH 7.4), for 10 min, permeabilized (0.5% Triton-X 100 in PBS) for 5 min, and then incubated with 100 nM rhodamine phalloidin for 30 min at room temperature in the dark. After counterstained with DAPI, the cells were visualized under a FLUOVIEW FV10i confocal microscope (Olympus, Tokyo, Japan). Statistical Analysis. All data are expressed as the mean ± SD from at least two independent experiments. Statistical significance was calculated by Student’s t test using Sigma plot software (Systat Software Inc., San Jose, CA, U.S.A.). A p value of
