Osthole Inhibits Insulin-like Growth Factor-1-Induced Epithelial to

Article pubs.acs.org/JAFC

Osthole Inhibits Insulin-like Growth Factor-1-Induced Epithelial to Mesenchymal Transition via the Inhibition of PI3K/Akt Signaling Pathway in Human Brain Cancer Cells Ying-Chao Lin,†,‡ Jia-Ching Lin,§ Chao-Ming Hung,∥,⊥ Yeh Chen,# Liang-Chih Liu,¶,△ Tin-Chang Chang,λ Jung-Yie Kao,§ Chi-Tang Ho,○ and Tzong-Der Way*,§,□,▲ †

Division of Neurosurgery, Buddhist Tzu Chi General Hospital, Taichung Branch, Taichung, Taiwan School of Medicine, Tzu Chi University, Hualien, Taiwan § Institute of Biochemistry, College of Life Science, National Chung Hsing University, Taichung, Taiwan ∥ Department of General Surgery, E-Da Hospital and ⊥School of Medicine, I-Shou University, Kaohsiung, Taiwan # Department of Biotechnology, Hungkuang University, Taichung, Taiwan ¶ Department of Surgery, China Medical University Hospital, Taichung, Taiwan △ School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan λ Department of Business Administration, Asia University, Taichung, Taiwan ○ Department of Food Science, Rutgers University, New Brunswick, New Jersey, United States □ Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan ▲ Department of Health and Nutrition Biotechnology, College of Health Science, Asia University, Taichung, Taiwan ‡

ABSTRACT: Glioblastoma multiforme (GBM) is one of the most lethal types of tumors and highly metastatic and invasive. The epithelial-to-mesenchymal transition (EMT) is the crucial step for cancer cells to initiate the metastasis and could be induced by many growth factors. In this study, we found that GBM8401 cells were converted to fibroblastic phenotype and the space between the cells became expanded in response to insulin-like growth factor-1 (IGF-1) treatment. Epithelial markers were downregulated and mesenchymal markers were upregulated simultaneously after IGF-1 treatment. Our results illustrate that IGF1 was able to induce EMT in GBM8401 cells. Osthole would reverse IGF-1-induced morphological changes, upregulated the expression of epithelial markers, and downregulated the expression of mesenchymal markers. Moreover, wound-healing assay also showed that osthole could inhibit IGF-1-induced migration of GBM8401 cells. By using dual-luciferase reporter assay and real-time PCR, we demonstrated that osthole inhibited IGF-1-induced EMT at the transcriptional level. Our study found that osthole decreased the phosphorylation of Akt and GSK3β and recovered the GSK3β bioactivity in inhibiting EMT transcription factor Snail and Twist expression. These results showed that osthole inhibited IGF-1-induced EMT by blocking PI3K/Akt pathway. We hope that osthole can be used in anticancer therapy and be a new therapeutic medicine for GBM in the future. KEYWORDS: glioblastoma multiforme, epithelial-to-mesenchymal transition, insulin-like growth factor-1, osthole, PI3K/Akt

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INTRODUCTION Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults. The current treatment standard for GBM includes surgical resection, radiotherapy, and chemotherapy with the DNA methylating agent temozolomide. Even with continuous improvements in the treatment and the most up-to-date chemotherapy, the dismal median survival time of GBM patients is less than 15 months.1,2 For this reason, it is vital to develop more effective targeted therapeutic agents to improve the overall survival of GBM patients. One of the major problems of GBM is tumor metastasis. Many aggressive tumors undergoing epithelial−mesenchymal transition (EMT) have been shown to enhance metastasis.3 The conversion of the immobile epithelial-like cancer cells to a motile mesenchymal-like phenotype requires alterations in morphology, cellular architecture, adhesion, and migration. EMT is characterized by the downregulation of epithelial markers and transcriptional induction of mesenchymal markers. Tran© 2014 American Chemical Society

scription factors like Twist, Slug, Snail, and ZEB1 lead to increased expression of mesenchymal markers (vimentin and Ncadherin) and concomitant decrease of epithelial markers (Ecadherin, ZO-1, and β-catenin).4 If we want to identify new targets for the prevention of metastasis, understanding the molecular mechanisms that drive EMT is important. Insulin-like growth factor (IGF) plays an important role in energy metabolism and tissue growth and development. Several studies have shown that IGF-1 or -2 increased cell proliferation and migration of various tumors in vitro.5 After binding of IGF-1 to the IGF-1 receptor (IGF-1R), tyrosine kinase on the cytoplasmic domain of IGF-1R activates multiple signaling pathways, including the RAS-RAF-MAPK and PI3K/Akt. Received: Revised: Accepted: Published: 5061

March 2, 2014 May 13, 2014 May 14, 2014 May 14, 2014 dx.doi.org/10.1021/jf501047g | J. Agric. Food Chem. 2014, 62, 5061−5071

Journal of Agricultural and Food Chemistry

Article

Figure 1. IGF-1-induced cell scattering and EMT in GBM8401 cells. GBM8401 cells were treated with DMSO (control) or increasing IGF-1 concentrations (100−300 ng/mL) for 24 h. (A) EMT was examined by phase contrast photomicrographs. (B) The cells were then harvested and lysed for the detection of N-cadherin, vimentin, E-cadherin, ZO-1, β-catenin, and β-actin. (C) The cells were then harvested and lysed for the detection of MMP-2, MMP-9, and β-actin. Western blot data presented are representative of those obtained in at least 3 separate experiments. Immunoblots were quantified, and the lower panel presents the average ± SD of three independent experiments. The value of the control cells was set to 1. (D) IGF-1induced cell motility was determined by measuring the closure of wound. Data were plotted by mean ± SD (n = 3). The closure distance of the control cells was set to 100 (**, p < 0.01;***, p < 0.001).

Compared to normal brain tissue, IGF-1R is overexpressed in malignant GBM.6 Recent studies suggested that IGF-1R activation, through autocrine or paracrine IGF-1 production, contributed to the proliferation and migration of GBM.7

Osthole, a coumarin derivative isolated from the fruit of Cnidium monnieri (L.) Cusson, has been widely used for the treatment of skin diseases and gynecopathy. Recently, osthole has taken considerable attention because of its broad spectrum of pharmacologic activities, including anti-inflammatory,8,9 anti5062

dx.doi.org/10.1021/jf501047g | J. Agric. Food Chem. 2014, 62, 5061−5071


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allergic,10 vasorelaxant,11 antidiabetic,12 and antitumor effects.13 In addition, current studies demonstrated that osthole had neuroprotective effects on animal models of central nervous system (CNS) diseases, such as brain ischemia models,14,15 and also in traumatic brain injury model.16 However, the anti-GBM ability of osthole remains largely unknown. A better understanding of the mechanisms behind the rapid growth of GBM may offer new therapeutic targets and improve outcome. IGF-1 activity has been closely examined in proliferative tissues for their relationship to change in cellular morphology associated with cancer progression. Therefore, the present study investigated whether IGF-1 could induce EMT in GBM cells. Since osthole has an ability to cross the blood−brain barrier and protect against brain injury, we further explore the potential of osthole in inhibiting the IGF-1-mediated EMT in GBM cells.

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medium. Before being scratched, the confluent monolayer was pretreated with the indicated concentrations of osthole for 2 h. A sterile 200 μL pipet tip was used to scratch the monolayers, followed by washing with PBS to remove cell debris. RPMI1640 containing 0.5% FBS, indicated concentrations of osthole, and 200 ng/mL IGF-1 were then added to each well for 24 h. A Nikon TE2000-U inverted microscope was used to measure ten random fields at 200× magnification. Data from three independent experiments were analyzed by GraphPad Prism 5 software. RNA Isolation and Quantitative Real-Time PCR. RNA isolation was performed as previously described.18 Briefly, total RNA was extracted according to the manufacturer’s protocol (Qiagen, Germantown, MD, USA). The extracted RNA was purified on an RNeasy purification column (Qiagen) and treated with DNase I according to the manufacturer’s protocol. Reverse transcriptase reactions were performed using a first-strand cDNA synthesis kit (Fermentas, Hanover, MD, USA). Quantitative real-time PCR was performed using StepOnePlus real-time PCR system (Applied Biosystems Inc., Foster City, CA, USA) on an ABI Prism 7500HT sequence detection system following the manufacturer’s instructions. In quantitative real-time PCR experiments, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was applied as an internal control. Primers used for E-cadherin were as follows: 5′-TGCTCTTCCAGGAACCTCTGTG-3′ (forward) and 5′GGTGACCACACTGATGACTCCTG-3′ (reverse). Reporter Construct Generation and Dual-Luciferase Reporter Assay. The reporter constructs were performed as previously described.18 For dual-luciferase reporter assay, 1 × 105 GBM8401 cells were plated onto 24-well plates 1 day before transfection and then transfected with reporter constructs by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After transient transfection, the GBM8401 cells were treated with osthole at various concentrations with or without IGF-1-stimulation. GBM8401 cells were lysed in the lysis buffer (Promega, Madison, WI, USA) and detected with dual luciferase assay kits (Promega). Tranfection. One day before transfection, 2 × 105 GBM8401 cells without serum were plated in a 6 cm dish. Cells were grown to 90% confluence and transfected on the following day by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and premixed plasmid DNA with OPTI-MEM (GIBCO, Carlsbad, CA, USA) for 5 min and then added to the dish. The transfection was completed after 24 h of incubation. Statistical Analysis. All values were expressed as mean ± SD. The independent Student’s t-test was used to compare the continuous variables between two groups, and the chi-squared test was applied for comparison of dichotomous variables. Asterisk indicates that the values were significantly different from the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

MATERIALS AND METHODS

Materials and Antibodies. Recombinant human IGF-1 was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Osthole and PD98059 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). LY294002 was purchased from Cayman Chemical Co. (Ann Arbor, Michigan, USA). Primary antibodies against IGF-1R, phosphoIGF-1R (Tyr 1135/1136), phospho-Akt (Ser 473), Akt, phospho-Erk1/ 2 (Thr 202/Tyr 204), Erk1/2, phospho-GSK3β (Ser 9), GSK3β, MMP2, MMP-9, N-cadherin, vimentin, ZO-1, β-catenin, Snail, and E-cadherin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for β-actin was purchased from Sigma Chemical Co (St. Louis, MO, USA). Antibodies for ZEB1 and Twist were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were also obtained from Santa Cruz Biotechnology, Inc. Cell Culture. HEK-293T cells were obtained from the American Type Culture Collection (Rockville, MD, USA). GBM8401 cells were obtained from Bioresources Collection and Research Center (Hsinchu, Taiwan). GBM8401 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin. Cell culture materials were obtained from Invitrogen (Burlington, Ontario, Canada). Morphology Observation. GBM8401 cells (2 × 104) were seeded in a 24-well plate and pretreated with the indicated concentrations of osthole for the appropriate time. IGF-1 was added to each well with indicated concentrations, and cells were then incubated for 24 h. A Nikon TE2000-U inverted microscope was used to take representative photographs at 200× magnification. Western Blot Analysis. Western blot was done as described previously.17 Briefly, cells (2 × 106) were treated with various agents as indicated in figure legends. After treatment, cells were placed on ice, washed with cold PBS, and lysed in the lysis buffer. Fifty micrograms of protein extract was loaded into sodium dodecyl sulfate polyacrylamide gels, and the separated proteins were transferred to nitrocellulose filters. The filters were probed with the appropriate primary antibody. Cell Viability Studies. To evaluate the cell viability of osthole in GBM8401 cells, MTT assay was employed. GBM8401 cells at a density of 1 × 104 cells per well were exposed to osthole for 24 h. MTT solution (stock concentration: 5 mg/mL in PBS) was diluted to 500 μg/mL. Each well was treated with 100 μL of MTT working solution and incubated at 37 °C for 2 h. The optical density was measured using an ELISA reader at 570 nm wavelength. Confocal Microscopy. After treatment, GBM8401 cells were fixed for 40 min with 4% paraformaldehyde in PBS at room temperature, permeabilized and blocked with 5% nonfat dry milk, 1% BSA, and 0.5% Triton X-100, and incubated overnight with anti-vimentin monoclonal antibody and then FITC-conjugated anti-mouse IgG antibody. DAPI staining was used to determine the nuclei. Cells were imaged with a Leica TCS SP2 Spectral Confocal System (Leica, Wetzlar, Germany). Wound Healing Assay. 4.5 × 105 GBM8401 cells were plated on a 6-well plate to form a confluent monolayer in serum-containing

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RESULTS IGF-1-Induced GBM8401 Cells Undergo EMT. To determine whether IGF-1 could initiate cell morphology changes, GBM8401 cells were treated with various concentrations (100−300 ng/mL) of IGF-1 for 24 h. GBM8401 cells maintained a classic cobblestone epithelial morphology; after exposure of GBM8401 cells to IGF-1, morphology was changed to fibroblast-like and the cell−cell contact was reduced (Figure 1A). GBM8401 cells were treated with various concentrations of IGF-1 for 24 h, and IGF-1 decreased the expression of epithelial phenotype markers, E-cadherin, ZO-1, and β-catenin in a dosedependent manner (Figure 1B). Inversely, IGF-1 increased the expression of mesenchymal markers N-cadherin and vimentin in a dose-dependent manner (Figure 1B). Our results indicated that IGF-1 induced EMT in GBM cells. We also determined whether IGF-1 regulated MMP-2 and MMP-9 expressions related to invasion and metastasis. The expression of MMP-2 and MMP-9 was increased after IGF-1 treatment for 24 h (Figure 1C). Moreover, IGF-1 induced an increase in cell migration (Figure 1D).

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Figure 2. IGF-1 induced phosphorylation of Akt and Erk1/2 and Snail and Twist expression in GBM8401 cells. GBM8401 cells were treated with DMSO (control) or increasing IGF-1 concentrations (100−300 ng/mL) for 10 min. (A) The cells were then harvested and lysed for the detection of IGF-1R, p-IGF-1R, and β-actin. (B) The cells were then harvested and lysed for the detection of p-Akt, Akt, p-GSK3β, p-Erk1/2, and β-actin. (C) GBM8401 cells were treated with DMSO (control) or increasing IGF-1 concentrations (100−300 ng/mL) for 24 h. The cells were then harvested and lysed for the detection of Snail, Twist, ZEB1, and β-actin. (D) GBM8401 cells were pretreated with 40 μM LY294002 or 20 μM PD98059 for 2 h prior to with or without IGF-1 stimulation (200 ng/mL) for 10 min. The cells were then harvested and lysed for the detection of p-Akt, Akt, p-GSK3β, p-Erk1/2, and β-actin. (E) BM8401 cells were pretreated with 40 μM LY294002 or 20 μM PD98059 for 2 h prior to with or without IGF-1 stimulation (200 ng/ mL) for 24 h. The cells were then harvested and lysed for the detection of Snail, Twist, and β-actin. Western blot data presented are representative of those obtained in at least 3 separate experiments. Immunoblots were quantified, and the value of the control cells was set to 1.

IGF-1-Induced EMT by Upregulating Snail and Twist in GBM8401 Cells. To investigate the ability of IGF-1-induced EMT through IGF-1R signaling, tyrosine phosphorylation of IGF-1R was examined in the presence of IGF-1. GBM8401 cells showed no detectable phosphorylation of IGF-1R when cells were cultured in a serum-free medium. In the presence of IGF-1, however, key tyrosine residues in the kinase domain of the βsubunit of IGF-1R became phosphorylated. There was no change

in the amount of total IGF-1R as determined by immunoblotting (Figure 2A). We next determined the effect of IGF-1 on the key signaling molecules downstream of IGF-1R, Akt and Erk1/2. IGF-1 stimulation resulted in increased phosphorylation of Akt and Erk1/2 in comparison to serum-free condition (Figure 2B). Interestingly, IGF-1 stimulation also resulted in increased phosphorylation of GSK3β, the downstream of Akt, in a dosedependent manner (Figure 2B). We next examined whether 5064



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Figure 3. Osthole blocked IGF-1-induced cell scattering and EMT in GBM8401 cells. (A) Chemical structure of osthole. (B) GBM8401 cells were treated with increasing osthole concentrations (10−100 μM) for 24 h. The cell viability was then determined using MTT assay. This experiment was repeated three times. The data represented the mean ± SD. (C) GBM8401 cells were pretreated with DMSO (control) or increasing osthole concentrations (20−80 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. (D) GBM8401 cells were incubated with DMSO (control) or 80 μM osthole for 1, 2, or 4 h prior to IGF-1 stimulation (200 ng/mL). EMT was examined by phase contrast photomicrographs. (E) GBM8401 cells were pretreated with DMSO (control) or increasing osthole concentrations (20−80 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. The cells were then harvested and lysed for the detection of N-cadherin, vimentin, E-cadherin, ZO-1, β-catenin, Snail, Twist, and β-actin. (F) GBM8401 cells were incubated with DMSO (control) or 80 μM osthole for 1, 2, or 4 h prior to IGF-1 stimulation (200 ng/mL). The cells were then harvested and lysed for the detection of N-cadherin, vimentin, E-cadherin, ZO-1, β-catenin, Snail, Twist, and β-actin. Western blot data presented are representative of those obtained in at least 3 separate experiments. Immunoblots were quantified, and the value of the control cells was set to 1.

EMT-inducing regulators, Snail, Twist, and Zeb1, were involved in IGF-1-induced EMT. GBM8401 cells were treated with different concentrations of IGF-1, and Western blotting analysis showed that IGF-1 increased both Snail and Twist expressions, but not the ZEB1 expression (Figure 2C). In order to further confirm whether Akt and Erk1/2 were involved in IGF-1mediated EMT, LY294002, a specific inhibitor of PI3K, and

PD98059, a specific inhibitor of MAPK, were used to inhibit IGF-1-induced phosphorylation of Akt and Erk1/2 in GBM8401 cells. The results showed that IGF-1-induced phosphorylation of Akt and Erk1/2 (Figure 2D) and upregulation of Snail and Twist (Figure 2E) were significantly inhibited by pretreatment with 40 μM LY294002 or 20 μM PD98059. Taken together, IGF-1 5065



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Figure 4. Osthole blocked IGF-1-induced cell migration in GBM8401 cells. (A) GBM8401 cells were pretreated with DMSO (control), 40 μM LY294002, or increasing osthole concentrations (20−80 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. Cells were fixed, permeabilized, and stained with anti-vimentin monoclonal antibody (green) and DAPI (blue). Cells were analyzed by confocal microscopy. (B) GBM8401 cells were pretreated with DMSO (control) or increasing osthole concentrations (20−80 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. The cells were then harvested and lysed for the detection of MMP-2, MMP-9, and β-actin. (C) GBM8401 cells were pretreated with DMSO (control) or 80 μM osthole for 1, 2, and 4 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. The cells were then harvested and lysed for the detection of MMP-2, MMP-9, and β-actin. Western blot data presented are representative of those obtained in at least 3 separate experiments. Immunoblots were quantified, and the value of the control cells was set to 1. (D) GBM8401 cells were pretreated with DMSO (control) or increasing osthole concentrations (20, 40 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. IGF-1-induced cell motility was determined by measuring the closure of wound. Data were plotted by mean ± SD (n = 3). The closure distance of the control cells was set to 100. Osthole significantly inhibited IGF-1-induced cell motility (*, p < 0.05).

with osthole prior to stimulation with IGF-1. To ensure that osthole blocked IGF-1-induced fibroblast-like morphology was not due to inhibition of proliferation, we tested the cytotoxicity of osthole on GBM8401 cells. As shown in Figure 3B, osthole had no significant cytotoxicity on GBM8401 cells. GBM8401 cells were pretreated with osthole (20−80 μM) for 2 h prior to stimulation with IGF-1 (200 ng/mL) for 24 h. Osthole blocked

induced phosphorylation of Akt and Erk1/2 and expression of Snail and Twist leading to EMT induction. Osthole Blocked IGF-1-Induced EMT in GBM8401 Cells. The activation of IGF-1 signaling significantly caused fibroblastlike morphology in GBM8401 cells. We next determined whether osthole (Figure 3A) could affect the IGF-1-induced fibroblast-like morphology, and GBM8401 cells were pretreated 5066



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Figure 5. Osthole blocked IGF-1-induced EMT at transcriptional level. (A) GBM8401 cells were pretreated with DMSO (control), 40 μM LY294002, or increasing osthole concentrations (20−80 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. Quantitative real-time PCR was performed by the StepOnePlus real-time PCR system and GAPDH was applied as an internal control. Data were plotted by mean ± SD (n = 3). The value of control was set to 1. Asterisk, values significantly different from the IGF-1 stimulation. *, p < 0.05; **, p < 0.01. (B) After cotransfection with pRL-CMV and pXP2-E-cadherin in GBM8401 cells, cells were pretreated with DMSO (control) or increasing osthole concentrations (20, 40 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. E-cadherin promoter activity was analyzed by luciferase reporter assays. Data were plotted by mean ± SD (n = 3). The value of control was set to 1. Asterisk, values significantly different from the IGF-1 stimulation. *, p < 0.05; **, p < 0.01. (C) Top: schematic representation of the wild-type (pXP2-E-cadherin) E-cadherin promoter construct. E1, E2, and E3 indicate the E-boxes. Bottom: promoter-activity assay. HEK-293T cells were cotransfected with a promoter construct and the indicated vector. All cells were cotransfected with a plasmid that expressed β-galactosidase, and the luciferase activity values were normalized to the β-galactosidase activity. After cotransfection, the cells were pretreated with DMSO (control) or increasing osthole concentrations (20, 40 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. For each promoter, all values were standardized to the luciferase activity of the control; asterisks indicate P < 0.05, compared with the control cells. The data are shown as the means ± SD (n = 3). (D) Top: schematic representation of the mutated-E-boxes (pXP2-E-cadherin (mut-E1E2E3)) E-cadherin promoter construct. E1, E2, and E3 indicate the E-boxes. Bottom: promoter activity assay in HEK-293T cells under different transfection conditions. A vector expressing βgalactosidase was cotransfected to ensure the transfection efficiency and normalize the luciferase activity values. After cotransfection, the cells were pretreated with DMSO (control) or increasing osthole concentrations (20, 40 μM) for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. For each promoter, all values were standardized to the luciferase activity of the control. The data are shown as the means ± SD (n = 3).

IGF-1-induced fibroblast-like morphology in a dose-dependent fashion (Figure 3C). Moreover, we pretreated GBM8401 cells with 80 μM osthole for varying periods of time from 1 to 4 h prior to IGF-1 addition. An increase in inhibition of IGF-1-induced fibroblast-like morphology was observed beginning at 1 h of osthole pretreatment (Figure 3D). To further identify whether inhibition of IGF-1-induced fibroblast-like morphology in osthole treated GBM8401 cells resulted from inhibition of EMT, we examined the expression of EMT related proteins in GBM8401 cells. Pretreated GBM8401 cells with osthole significantly suppressed the IGF-1-induced upregulation of Ncadherin, vimentin, Snail, and Twist and downregulation of ZO-1 and β-catenin in a dose- and time-dependent manner (Figure 3E,F). Osthole Blocked IGF-1-Induced Cell Migration by Downregulating MMP-2, and MMP-9 in GBM8401 Cells. To further examine that osthole suppressed the IGF-1-induced upregulation of vimentin, GBM8401 cells were imaged with

confocal microscopy. As shown in Figure 4A, confocal microscopy with vimentin antibodies indicated that IGF-1 treatment resulted in upregulation of vimentin. Pretreatment with various concentrations of osthole for 2 h significantly suppressed the IGF-1-induced upregulation of vimentin in a dose-dependent manner (Figure 4A). IGF-1 has been shown previously to stimulate protease activity in many cell types.19 To determine whether IGF-1 could stimulate metalloproteinase activity, GBM8401 cells were treated with 200 ng/mL IGF-1. IGF-1 stimulation resulted in increased expression of MMP-2 and MMP-9 compared to serum-free conditions (Figure 4B,C). Pretreatment with osthole significantly suppressed the IGF-1induced upregulation of MMP-2 and MMP-9 in a dose- and time-dependent manner (Figure 4B,C). We next examined whether osthole inhibited IGF-1-induced cell migration by measuring the wound closure. Compared to GBM8401 cells treated with DMSO (control), IGF-1 significantly induced cell 5067



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Figure 6. Osthole blocked IGF-1-induced Akt pathway in a time-dependent manner. (A) GBM8401 cells were preincubated in the presence or the absence of 80 μM osthole for 2 h prior to IGF-1 stimulation (200 ng/mL) for various times. The cells were then harvested and lysed for the detection of p-Akt, Akt, p-GSK3β, p-Erk1/2, and β-actin. (B) GBM8401 cells were pretreated with 80 μM osthole, or 40 μM LY294002, or a combination of 40 μM LY294002 and 80 μM osthole for 2 h and then treated with IGF-1 for 10 min. The cells were then harvested and lysed for the detection of p-Akt, pGSK3β, and β-actin. GBM8401 cells were pretreated with 80 μM osthole, or 40 μM LY294002, or a combination of 40 μM LY294002 and 80 μM osthole for 2 h and then treated with IGF-1 for 24 h. (C) The cells were then harvested and lysed for the detection of Snail, Twist, and β-actin. (D) The cells were then harvested and lysed for the detection of N-cadherin, vimentin, β-catenin, ZO-1, and β-actin. (E) The cells were then harvested and lysed for the detection of MMP-2, MMP-9, and β-actin. (F) Cell motility was determined by measuring the closure of wound. Data were plotted by mean ± SD (n = 3). Osthole or a combination of LY294002 and osthole significantly inhibited IGF-1-induced cell motility (**, p < 0.01;***, p < 0.001). (G) GBM8401 cells were transfected with 50 nmol/L CA-Akt or mock. Twenty-four hours after transfection, preincubation was carried out in the presence or the absence of 80 μM osthole for 2 h prior to IGF-1 stimulation (200 ng/mL) for 24 h. The cells were then harvested and lysed for the detection of Ncadherin, ZO-1, and β-actin. Western blot data presented are representative of those obtained in at least three separate experiments. The values below the figures represent change in protein expression normalized to β-actin.

migration. Significantly, the IGF-1-induced cell migration was inhibited in the presence of osthole (Figure 4D). Osthole Suppressed IGF-1 Inducible E-Cadherin Transcriptional Downregulation. We next determined whether pretreatment with osthole could inhibit IGF-1-induced downregulation of E-cadherin through transcriptional level. Real-time PCR analysis was performed to assess the effect of osthole on Ecadherin mRNA expression. As shown in Figure 5A, IGF-1induced downregulation of E-cadherin mRNA was restored by

pretreatment with osthole. Our luciferase assay indicated that IGF-1 inhibited E-cadherin promoter activity, and pretreatment with osthole significantly restored the IGF-1-repressed Ecadherin promoter activity in a dose-dependent manner (Figure 5B). Figure 2C indicates that IGF-1 induced Snail and Twist expression. Interestingly, Snail and Twist bind to the same elements in the E-cadherin promoter: three E-boxes with a core 5′-CACCTG-3′ sequence. We next investigated whether osthole suppressed the binding of Snail and Twist to these E-boxes and 5068



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repressed the E-cadherin promoter. Promoter activity was analyzed by transfection of E-cadherin promoter containing three E-boxes constructed into HEK-293T cells. Our promoteractivity assay showed that IGF-1 inhibited E-cadherin promoter activity. Pretreatment with various concentrations of osthole restored the IGF-1-repressed E-cadherin promoter activity (Figure 5C). As shown in Figure 5D, mutation of all three Eboxes abrogated repression by IGF-1. Pretreatment with various concentrations of osthole was not able to restore the E-boxesmutated E-cadherin promoter activity. These data suggest that osthole inhibits IGF-1-mediated EMT through transcriptional level. Osthole Inhibited IGF-1-Induced EMT through Inhibiting PI3K/Akt Signaling Pathway. To determine whether osthole inhibited the key signaling molecules downstream of IGF-1R, Akt and Erk1/2, the phosphorylation of Akt and Erk1/2 was examined at various time points after adding IGF-1 (200 ng/ mL) to GBM8401 cells. The phosphorylation of Akt and Erk1/2 was increased at 15 min after stimulation, and this effect lasted for at least 120 min (Figure 6A). Pretreatment with osthole (80 μM) for 2 h significantly suppressed the IGF-β-induced phosphorylation of Akt and GSK3β (Figure 6A). However, pretreatment with osthole was not able to suppress the IGF-β-induced phosphorylation of Erk1/2 (Figure 6A). We next investigated whether Akt was involved in osthole inhibited IGF-1-mediated EMT; PI3K inhibitor LY294002 was used. Our study showed that IGF-1-induced phosphorylation of Akt and GSK3β (Figure 6B), upregulation of Snail and Twist (Figure 6C), upregulation of N-cadherin and vimentin and downregulation of β-catenin and ZO-1 (Figure 6D), and upregulation of MMP-2 and MMP-9 (Figure 6E) were significantly inhibited by pretreatment with 40 μM LY294002. Interestingly, in the cotreatment of LY294002 (40 μM) and osthole (80 μM), IGF-1-induced phosphorylation of Akt and GSK3β (Figure 6B), upregulation of Snail and Twist (Figure 6C), upregulation of N-cadherin and vimentin and downregulation of β-catenin and ZO-1 (Figure 6D), and upregulation of MMP-2 and MMP-9 (Figure 6E) were more potent than pretreatment with LY294002 or osthole alone. We further confirmed whether Akt was involved in osthole inhibited IGF-1-mediated cell migration. Figure 6F showed that IGF-1induced cell migration was significantly inhibited by pretreatment with 40 μM LY294002. In the cotreatment of LY294002 (40 μM) and osthole (80 μM), IGF-1-induced cell migration was more potent than pretreatment with LY294002 or osthole alone (Figure 6F). To assess whether Akt was involved in osthole inhibited IGF-1-mediated EMT, we performed Akt-overexpression experiment using constitutively active Akt (CAAkt). The result showed that CA-Akt abolished the ability of osthole to suppress IGF-1-induced EMT (Figure 6G). Taken together, our data suggest that the inhibition of PI3K/Akt signaling pathway is involved in osthole inhibited IGF-1mediated EMT, and cell migration in GBM8401 cells.

The EMT is crucial for the migration and invasion of many epithelial tumors. IGF-1 stimulation has been shown to promote dramatic neomorphic effects of an EMT and enhanced cell migration and invasion. For example, Graham et al. have clearly shown that aberrant expression of ZEB1 occurs in part due to IGF-1, and the activated ZEB1 is then able to promote epithelial prostate cancer cells to exhibit a mesenchymal phenotype.23 Kim et al. found that overexpression of a constitutively activated IGF1R was sufficient to cause human mammary epithelial cells to undergo an EMT which was associated with dramatically increased migration and invasion.24 Moreover, overexpression of IGFBP4 in U343 glioma cells resulted in upregulation of molecules involved in tumor growth, EMT, and invasion.25 Herein, we showed that IGF-1 treatment caused GBM8401 cells to lose their polygonal appearance and cell−cell contacts leading to the acquisition of an elongated, spindle-shaped morphology, suggesting that GBM8401 cells underwent EMT. Taken together, these data indicate that IGF-1 can initiate EMT in cancer cells and promote tumor cell metastasis. Moreover, targeting IGF-1 signaling in GBM may be a promising anticancer strategy. There are several zinc finger transcription factors, including Snail, Twist, and ZEB1, which have been described as E-cadherin repressors. Herein, our Western blotting analysis showed that IGF-1 increased both the expressions of Snail and Twist, but not the ZEB1 expression. Mikheeva et al. revealed that Twist promoted invasion through mesenchymal changes in GBM.26 Zhang et al. indicated that Snail promoted the migratory and invasive properties of glioma cells.27 Taken together, these data show that expression of Snail and Twist is characteristic of mesenchymal tumor areas of GBM, suggesting that similar mechanisms involved in the EMT in epithelial neoplasms played a role in mesenchymal differentiation in GBM. Following binding of IGF-1 to the extracellular subunits of the IGF-1R, it leads to the activation of at least 2 main pathways: first via RAS, RAF, and Erk1/2, and second via the PI3K and Akt signaling pathway. Our data suggest that Erk1/2 and Akt are upstream factors of Snail and Twist activation in GBM cells. IGF1 has been shown to activate the Erk1/2 and Akt signaling pathways in various cancer cells, and we showed in GBM8401 cells that Snail and Twist expressions were Erk1/2 and Akt dependent. Pharmacologic inhibition of Erk1/2 and Akt signaling has been shown to decrease invasion or inhibit specific biochemical changes consistent with EMT.28,29 Our results are consistent with previous reports, suggesting that Erk1/2- and Akt-dependent pathways are sufficient to maintain the mesenchymal phenotype. Recent studies showed that osthole regulates the proliferation, apoptosis, and invasion in many cancer cells via multiple signaling pathways.30,31 Moreover, osthole has essential roles in brain functions by protecting neurons, suggesting that osthole could penetrate the blood−brain barrier for the chemotherapy drugs of brain tumors.14−16 In the present study, we first evaluated the effects of osthole on IGF-1 induced EMT in GBM8401 cells. The present study showed that IGF-1 induced EMT in GBM8401 cells and this process could be effectively blocked by osthole. Pretreatment with osthole blocks IGF-1induced EMT, however, how osthole affects these changes remain to be determined. To further delineate the molecular mechanism of osthole blocking IGF-1-induced EMT, related pathways were investigated. In the study, both Erk1/2 and PI3K/Akt signaling pathways were investigated in osthole-treated GBM8401 cells.

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DISCUSSION Recent studies indicated an important role for the IGF signaling pathway in the proliferation, progression, and antiapoptosis of GBM cells. For example, treatment of glioblastoma cells with IGF-1 increased cellular proliferation and migration.20 Inhibition of IGF-1R by tyrosine kinase inhibitor inhibited GBM cell growth.21 IGF-1R inhibition contributed to the growth suppression of primary cell lines derived from human highgrade gliomas.22 5069



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lipopolysaccharide-induced acute lung injury in mice by preventing down-regulation of angiotensin-converting enzyme 2. Eur. J. Pharm. Sci. 2013, 48, 819−824. (9) Nakamura, T.; Kodama, N.; Arai, Y.; Kumamoto, T.; Higuchi, Y.; Chaichantipyuth, C.; Ishikawa, T.; Ueno, K.; Yano, S. Inhibitory effect of oxycoumarins isolated from the Thai medicinal plant Clausena guillauminii on the inflammation mediators, iNOS, TNF-alpha, and COX-2 expression in mouse macrophage RAW 264.7. J. Nat. Med. 2009, 63, 21−27. (10) Matsuda, H.; Tomohiro, N.; Ido, Y.; Kubo, M. Anti-allergic effects of cnidii monnieri fructus (dried fruits of Cnidium monnieri) and its major component, osthol. Biol. Pharm. Bull. 2002, 25, 809−812. (11) Yao, L.; Lu, P.; Li, Y.; Yang, L.; Feng, H.; Huang, Y.; Zhang, D.; Chen, J.; Zhu, D. Osthole relaxes pulmonary arteries through endothelial phosphatidylinositol 3-kinase/Akt-eNOS-NO signaling pathway in rats. Eur. J. Pharmacol. 2013, 699, 23−32. (12) Liang, H. J.; Suk, F. M.; Wang, C. K.; Hung, L. F.; Liu, D. Z.; Chen, N. Q.; Chen, Y. C.; Chang, C. C.; Liang, Y. C. Osthole, a potential antidiabetic agent, alleviates hyperglycemia in db/db mice. Chem.-Biol. Interact. 2009, 181, 309−315. (13) Hung, C. M.; Kuo, D. H.; Chou, C. H.; Su, Y. C.; Ho, C. T.; Way, T. D. Osthole suppresses hepatocyte growth factor (HGF)-induced epithelial-mesenchymal transition via repression of the c-Met/Akt/ mTOR pathway in human breast cancer cells. J. Agric. Food Chem. 2011, 59, 9683−9690. (14) Chao, X.; Zhou, J.; Chen, T.; Liu, W.; Dong, W.; Qu, Y.; Jiang, X.; Ji, X.; Zhen, H.; Fei, Z. Neuroprotective effect of osthole against acute ischemic stroke on middle cerebral ischemia occlusion in rats. Brain Res. 2010, 1363, 206−211. (15) Chen, T.; Liu, W.; Chao, X.; Qu, Y.; Zhang, L.; Luo, P.; Xie, K.; Huo, J.; Fei, Z. Neuroprotective effect of osthole against oxygen and glucose deprivation in rat cortical neurons: involvement of mitogenactivated protein kinase pathway. Neuroscience 2011, 183, 203−211. (16) He, Y.; Qu, S.; Wang, J.; He, X.; Lin, W.; Zhen, H.; Zhang, X. Neuroprotective effects of osthole pretreatment against traumatic brain injury in rats. Brain Res. 2012, 1433, 127−136. (17) Hsu, S. C.; Lin, J. H.; Weng, S. W.; Chueh, F. S.; Yu, C. C.; Lu, K. W.; Wood, W. G.; Chung, J. G. Crude extract of Rheum palmatum inhibitsmigration and invasion of U-2 OS human osteosarcoma cells by suppression of matrix metalloproteinase-2 and -9. BioMedicine 2013, 3, 120−129. (18) Way, T. D.; Huang, J. T.; Chou, C. H.; Huang, C. H.; Yang, M. H.; Ho, C. T. Emodin represses TWIST1-induced epithelial-mesenchymal transitions in head and neck squamous cell carcinoma cells by inhibiting the β-catenin and Akt pathways. Eur. J. Cancer 2014, 50, 366−378. (19) Vincent-Salomon, A.; Thiery, J. P. Host microenvironment in breast cancer development: epithelial-mesenchymal transition in breast cancer development. Breast Cancer Res. 2003, 5, 101−106. (20) Schlenska-Lange, A.; Knüpfer, H.; Lange, T. J.; Kiess, W.; Knüpfer, M. Cell proliferation and migration in glioblastoma multiforme cell lines are influenced by insulin like growth factor I in vitro. Anticancer Res. 2008, 28, 1055−1060. (21) Yin, S.; Girnita, A.; Strömberg, T.; Khan, Z.; Andersson, S.; Zheng, H.; Ericsson, C.; Axelson, M.; Nistér, M.; Larsson, O.; Ekström, T. J.; Girnita, L. Targeting the insulin-like growth factor-1 receptor by picropodophyllin as a treatment option for glioblastoma. NeuroOncology 2010, 12, 19−27. (22) Carapancea, M.; Cosaceanu, D.; Budiu, R.; Kwiecinska, A.; Tataranu, L.; Ciubotaru, V.; Alexandru, O.; Banita, M.; Pisoschi, C.; Bäcklund, M. L.; Lewensohn, R.; Dricu, A. Dual targeting of IGF-1R and PDGFR inhibits proliferation in high-grade gliomas cells and induces radiosensitivity in JNK-1 expressing cells. J. Neurooncol. 2007, 85, 245− 254. (23) Graham, T. R.; Zhau, H. E.; Odero-Marah, V. A.; Osunkoya, A. O.; Kimbro, K. S.; Tighiouart, M.; Liu, T.; Simons, J. W.; O’Regan, R. M. Insulin-like growth factor-I-dependent up-regulation of ZEB1 drives epithelial-to-mesenchymal transition in human prostate cancer cells. Cancer Res. 2008, 68, 2479−2488.

GBM8401 cells were pretreated with osthole and showed significant decrease of phospho-Akt but no obvious decrease of phospho-Erk1/2. Our previous studies showed that osthole suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating the Akt/mTOR pathway31 and suppresses the hepatocyte growth factor (HGF)induced epithelial−mesenchymal transition via repression of the c-Met/Akt/mTOR pathway in human breast cancer cells.13 Ding et al. revealed that osthole inhibited the proliferation, promoted apoptosis, and inhibited cell migration/invasion in vitro in glioma cells through inhibiting both PI3K/Akt and MAPK signaling pathways.32 Xu et al. showed that osthole induced G2/ M arrest and apoptosis in lung cancer A549 cells by modulating the PI3K/Akt pathway.33 Based on these findings, osthole may be an ideal inhibitor for PI3K/Akt signaling pathway.

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AUTHOR INFORMATION

Corresponding Author

*Department of Biological Science and Technology, College of Life Sciences, China Medical University, No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402. Tel: +886-4-2205-3366, ext 2509. Fax: +886-4-2203-1075. E-mail: [email protected]. Funding

This study was supported by the National Science Council of the Republic of China, Grant NSC 101-2320-B-039-031-MY3. Thanks are also due to support (in part) by a grant from the Department of Health (Taiwan), China Medical University Hospital Cancer Research Center of Excellence (DOH100-TDC-111-005), and a grant from China Medical University (CMU101-N2-03). The authors would like to thank Prof. Mien-Chie Hung for generously providing the cDNA of CA-Akt. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED BBB, blood−brain barrier; EMT, epithelial−mesenchymal transition; MMPs, matrix metalloproteinases; IGF-1, insulinlike growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; IGFBP, IGF binding protein; IRS, insulin receptor substrate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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REFERENCES

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Osthole Inhibits Insulin-like Growth Factor-1-Induced Epithelial to

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