Marchantin C a Potential Anti-Invasion Agent in Glioma Cells

www.landesbioscience.com Cancer Biology & Therapy 33

Cancer Biology & Therapy 9:1, 33-39; January 1, 2010; © 2010 Landes Bioscience

ReseaRCh PaPeR ReseaRCh PaPeR

*Correspondence to: Gang Li and hongxiang Lou; email: [email protected] and [email protected]: 07/19/09; Revised: 10/02/09; accepted: 10/09/09Previously published online: www.landesbioscience.com/journals/cbt/article/10279

Introduction

Malignant gliomas are the most common primary brain tumors. The median life expectancy for patients with glioblastoma multiforme is approximately 14 mo, with no more than 5% of patients alive 5 y after diagnosis1 despite advanced multiple treatments. The highly invasive nature of glioma cells is a key problem in clinical treatment failure and tumor recurrence.2,3 Although systemic metastases are relatively rare, local invasion of single tumor cells to adjacent and distant brain structures is a hallmark of malignant glioma. During the invasion pro-cess cancer cells make changes in several morphologic features, such as cell polarization, filopodia formation, spreading, and migration,4 which are possible regulating points between the static stage and the metastatic stage of a cancer cell. The new tumor suppressors and candidate tumor inhibitory genes are involved in these processes at different steps, and may therefore provide valuable therapeutic strategies to prevent glioma from metastasis.

Macrocyclic bisbibenzyls, a large family of phenolic com-pounds belonging to stilbenoids,5 is a class of characteristic components from liverworts. More and more attention is being paid to them because of their wide range of biological activities including cytotoxicity and antibacterial and antifungal activity.6,7

Cancer cell migration is a leading cause of tumor recurrence and treatment failure. Previously, we reported that marchantin C exhibited promising antitumor activity by inducing microtubule depolymerization and apoptosis. In the present study, we investigated the effect of marchantin C on inhibition of migration in T98G and U87 cells. The scratch-induced migration, Boyden chamber and cell invasion assays were applied to determine that the migrating capacity and invasiveness of these glioma cell lines were inhibited when exposed to marchantin C at a low concentration. There are no obvious signs of apoptosis with this dose. Western blot analyses confirmed that MMP-2, a key role in cancer cell migration, was reduced after incubation with marchantin C in both glioma cell lines. In addition, signaling pathway investigations demonstrated that eRK/MaPK might be involved in MMP-2 downregulation, rather than the aKT/PI3K or JaK/sTaT3 pathways. Moreover, marchantin C potently suppressed angiogenesis activity in vivo by CaM assay. This is the first study to demonstrate that marchantin C can inhibit glioma cell migration and invasiveness.

Marchantin Ca potential anti-invasion agent in glioma cells

Jie shen,1 Gang Li,1,* Qinglin Liu,1 Qiaowei he,1 Jinhai Gu,2 Yanqiu shi3 and hongxiang Lou4,*

1Department of Neurosurgery; Qilu hospital of shandong University; Jinan, China; 2Ningxia Key Laboratory of Craniocerebral Diseases; Ningxia Medical University; Yinchuan, China; 3Centre of New Drugs evaluation of shandong University; Jinan, China; 4school of Pharmaceutical sciences; shandong University; Jinan, China

Key words: marchantin C, glioma, invasion, MAPK, PI3K, STAT3, CAM

Abbreviations: MAPK, mitogen-activated protein kinases; PI3K, phosphoinositide 3 kinase; STAT3, signal transducer and activator of transcription; CAM, chicken embryo chorioallantoic membrane; MMP, matrix metalloproteinase; VEGF, vascular

endothelial growth factor; PBS, phosphate-buffered saline

Marchantin C, a macrocyclic bisbibenzyl initially found in Marchantiophyta by a Japanese group, has been extensively studied. It has been demonstrated to exhibit multiple pharmaco-logical activities such as antimicrobial activity against the Gram-positive bacterium Bacillus subtilis,6 antioxidant activity to free radicals,5 and anti-tumor activity against A172 glioma cells.8 Recently, we found that marchantin C abrogated microtubule dynamics and mitotic spindle formation, resulting in cell cycle arrest at G

2/M phase and eventually leading to cell death both in

vitro and in vivo.9 However, it is unknown whether macrocyclic bisbibenzyl family compounds have anti-metastatic properties in cancer cells.

In the present study, we provide evidence to confirm that glioma cell invasion might be affected by marchantin C in a variety of experiments. Furthermore, we determined that march-antin C-mediated suppression of glioma cell invasion likely involves the ERK/MAPK signaling pathway instead of PI3K/AKT r JAK/STAT3 signaling pathways. We also observed that matrix metalloproteinase (MMP)-2 expression was reduced when glioma cells were exposed to marchantin C, as was the anti-angiogenesis activity as determined by CAM assay. Thus, our study might provide valuable insights into how marchantin C might be a potential antitumor agent by inhibiting invasive-ness in the brain tumor.

34 Cancer Biology & Therapy Volume 9 Issue 1

assay, cell migration ability was calculated as the ratio of recovery area in marchantin C treated groups to that in control groups. The migration rates of both T98G and U87 cells were decreased after treatment with marchantin C for 12 and 24 h. As shown in Figure 2A, the inhibition was both time- and dose-dependent, with maximum inhibition of 69.3% in T98G cells and 71.2% in U87 cells. In the Boyden chamber assay, a significant inhibition of glioma cell migration in the presence of marchantin C as com-pared to the control group was detected, which supported the results obtained from the scratch-induced assay (Fig. 2B). These data confirmed that marchantin C could decrease the migra-tion ability of glioma cells in both a two- and three-dimensional manner.

Marchantin C decreases invasion ability in glioma cells. Given that marchantin C plays an important role in migration, we further examined the effect of marchantin C on glioma cell inva-sion. An invasion system was set up as described in Materials and Methods and the solution of marchantin C was added into the lower wells at final concentration of 6, 8 or 10 μM. The invasive-ness of T98G and U87 cells was significantly decreased in a dose-response relationship as compared to untreated cells (Fig. 3A), which was consistent with the data shown in migration assays.

Results

Marchantin C inhibits the proliferation of U87 and T98G glioma cells. Cell proliferation was determined by the MTT assay in the presence of different concentrations of marchantin C at different time points. As shown in Figure 1A, marchantin C treatment caused potent growth inhibition in the two cell lines in a dose- and time-dependent manner. At the concentration of 12 μM, the viability of both cell lines was significantly decreased. Cell-counting demonstrated that marchantin C exhibited a minor effect on the viability of glioma cells at lower concentra-tions, with inhibition of 2.8, 4.9 and 9.0% in U87 cells and 3.0, 7.6 and 12.1% in T98G cells at the concentration of 6, 8 and 10 μM, respectively (Fig. 1B). As treatment with no more than 10 μM marchantin C resulted in suppression of cell proliferation and exhibited no major signs of apoptosis, this concentration was used for all further analysis.

Marchantin C decreases migration ability in glioma cells. To determine whether marchantin C plays a role in glioma cell migration, scratch-induced migration assays and Boyden cham-ber assays were performed in the absence or presence of different doses of marchantin C over a 24-h period. In the scratch-induced

Figure 1. evaluation of the cell viability of T98G and U87 glioma cell lines treated with marchantin C for 24 h. (a) T98G and U87 cells were incubated with marchantin C at concentration of 6, 8, 10 μM for 24 h and the number of surviving cells was determined using MTT assay as described in Materials and Methods. (B) Trypan Blue exclusion was used to assess the viability of T98G and U87 cells exposed to marchantin C at the proper concentration. The experiments were performed in triplicate and data are presented as means ± sD. * and ** indicate means that are significantly different when compared to the control group with 0.01 < p < 0.05 and p < 0.01, respectively.


of marchantin C in vitro and in vivo. Our results illustrated that the glioma cells did not show any signs of apoptosis in treatment with low concentrations of no more than 10 μM marchantin C. However, the inhibitory effect of marchantin C on the migration and invasion of glioma cells was striking. Whereas the mech-anism by which marchantin C reduces glioma mobility is not completely elucidated, we hypothesize that MMP-2 alteration may be involved.

Cancer cell invasion involves multiple steps, including attach-ment to barrier matrix, basement membrane degradation, and migration to a new place. In previous studies, a strong association between the invasive behavior of gliomas and expression of differ-ent metalloproteases had been reported.11,12 An essential part of invasion and metastasis, MMPs are responsible for degradation of basement membrane. As shown in Figure 3B and D, western blots showed that treatment of glioma cells with marchantin C for 24 h exhibited a significantly decreased expression of MMP-2.

Several pathways have been identified that lead to activa-tion of MMP-2. Recently, some groups reported that MMP-2, directly regulated by STAT3 signaling in human melanoma brain metastases, was a target gene of STAT3.13 In another

MMP-2, a well-known factor involved in tumor invasion, is capable of degrading the extracellular matrix and basement mem-branes. Therefore, we determined the protein level of MMP-2 by western blot. As shown in Figure 3B and D, a significant decrease in MMP-2 protein was observed after 24 h of exposure to marchantin C, likely illustrat-ing the mechanism by which marchantin C decreases glioma cell invasiveness.

Marchantin C inhibits phosphoryla-tion of ERK/MAPK signaling pathway. To determine which signal transduction pathway is involved in cell invasion and decreasing MMP2 expression, the protein levels of MAPK, PI3K and STAT3, together with their phosphorylation status, were determined by western blot. As shown in Figure 4, the levels of p-ERK1/2 in cells treated with marchantin C were signifi-cantly downregulated in a dose-dependent manner when compared to those in the con-trol groups. p-AKT and p-STAT3 protein levels had little changes. These results sug-gest that the ERK/MAPK signaling path-way may be involved in the downregulation of MMP2 mediated by marchantin C in the glioma cells.

Marchantin C inhibits angiogenesis in the CAM assay. It is well known that neovascularization is a major contributing factor in both tumor growth and metasta-sis. To explore whether marchantin C has anti-angiogenic potency in vivo, we used a chicken embryo chorioallantoic membrane (CAM) angiogenesis model. After 48 h of incubation with march-antin C, macroscopic findings demonstrated numerous allantoic vessels developed around the gelatin sponge in a spoked pattern in control groups, while the number of vessels and the vessel branch points were downregulated in a dose-dependent manner in marchantin C treated groups. We observed a maximum inhi-bition of 55.9% at 10 μM (Fig. 5). These data indicated a strong inhibitory effect of marchantin C on spontaneous angiogenesis.

Discussion

Our results have shown that marchantin C had a direct effect on the invasive potential of U87 and T98G glioma cells and have demonstrated some of the mechanisms involved.

Marchantin C is naturally isolated from liverworts Marchantia polymorpha L., Ptagiochasm intermedium L. and Asterella angusta.10 Studies and clinical trials have demonstrated that marchantin C has antibacterial, antifungal, and antioxidant properties,6,7 as well as suppression of the growth of several cancer cells, such as A172, K562 and HepG2.9 In the present study, we focused our main scope on the anti-invasiveness and anti-angiogenic activity

Figure 2. Migration assay of T98G and U87 glioma cells in the presence or absence of marchan-tin C as described in Materials and Method. T98G cells and U87 cells (a) migrated from scratch. Recovery of each denuded area was quantified by densitometric analyses relative to that of the control which was set at 100% as shown in the graph. Boyden chamber analysis on T98G and U87 cells (B). Results were expressed as the ratio of the number of migration cells to that of control. each data was obtained from three independent experiments and presented as means ± sD (*0.01 < p < 0.05; **p < 0.01).


the ability to metastasize.16 In the previous study, combretastatin A-4 phosphate (CA-4-P), a tubulin-depolymerising agent struc-turally related to colchicines, was proved to be a successful tumor vascular targeting agent.17 Marchantin C, previously proved to directly inhibit tubulin polymerization,9 consists of two bibenzyl skeletons linked with two ether bonds and its structure is similar to a dihydrated dimer of combretastatin A-4 (CA-4). Based on the similar structure and characteristics, we hypothesized that march-antin C may also have an anti-angiogenesis activity. In vivo CAM assays were performed and the results confirmed that marchantin C exhibited a potent inhibitive effect on spontaneous angiogen-esis in a dose-dependent manner, with a maximum inhibition of 55.9% (Fig. 5). A number of growth factors have been identified as potential regulators of angiogenesis, such as vascular endothe-lial growth factor (VEGF), basic fibroblast growth factor (TGFα), TGFβ, tumor necrosis factor, platelet-derived endothelial growth factor, and placenta growth factor.18-20 Accumulating evidence demonstrates that inhibition of VEGF expression is an effective

study, MMP-2 was involved in glioma invasion via the Ras/Raf/MEK-dependent pathway.14 Moreover, a proposed link-age between PI3K signaling and MMP activity in gliomas has been confirmed.15 However, the signaling pathways by which marchantin C regulates MMP-2 expression in glioma cells are not fully characterized. Subsequently, we analyzed the protein levels of STAT3, ERK1/2 and AKT, together with their phos-phorylation status, by western blot assays. As shown in Figure 4, the phosphorylation of ERK1/2 expression was significantly decreased after 24 h exposure to marchantin C, while the levels of STAT3 and AKT phosphorylation had little change compared to that in control groups. This is the first report that marchantin C-mediated suppression of invasion of glioma cells may affect MMP production predominantly by inhibiting the MAPK/ERK signaling pathway, rather than PI3K/AKT or JAK/STAT3 pathway.

Angiogenesis is a vital process for the progression of a neo-plasm from a small localized tumor to an enlarging tumor with

Figure 3. Marchantin C inhibits glioma cells invasion. (a) Transwell chamber with Matrigel-precoated membrane filter inserts were used to measure invasiveness as described in Material and Methods. The glioma cells that migrated through the membrane were stained, and representative fields were photographed. Original magnification: x200, Black bar: 50 μm. (B) Marchantin C decreased the protein expression of MMP-2. (C) Invasion was quantified by counting the cells. The data are the average of the number of cells/field of six random fields in three independent experiments (*p < 0.01). (D) The histogram represented the ratio of MMP-2 expression of each treatment group to the control group (*p < 0.01).


Academy of Sciences (Shanghai, China). The cells were routinely incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, Grand Island, NY USA) and Eagle’s minimum essen-tial medium (EMEM, GIBCO, Grand Island, NY USA) supple-mented with 10% fetal bovine serum (TBD, TianJing, China) with 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified air with 5% CO

2 at 37°C.

MTT assay. MTT was adopted to detect the inhibitory potency of marchantin C in glioma cell proliferation. U87 and T98G cells were plated in 96-well plates at a density of 8 x 103 cells per well and 5 x 103 cells per well, respectively. After 24 h incubation, the cells were treated with marchantin C of varying concentrations (0, 6, 8, 12, 16 and 20 μM) and incubated in growth medium for 24, 48 or 72 h. After removing the medium, 20 μL MTT [5 mg/mL, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; Sigma, USA] was added to each well and incubated at 37°C for 4 h. After the addition of 150 μL DMSO to each well, the optical density value at 540 nm (OD540) was measured using a microplate reader (Bio-Rad).

Cell viability analysis. Trypan dye exclusion assay was used to evaluate cell viability. U87 and T98G glioma cells were seeded at

strategy for brain cancer therapy. Yunker et al. report that SPARC modulates glioma growth by sup-pressing tumor vascularity through suppression of VEGF expression and secretion.21 Slongl et al. report that targeting VEGF signaling may rep-resent a new therapeutic option in the treatment of medulloblastoma.22 Cantarella et al. suggested that TRAIL inhibits angiogenesis stimu-lated by VEGF expression in human glioblastoma cells.23 In the present study, we found that there was a modest change in VEGF expression in these two glioma cell lines upon exposure to marchantin C (data not shown), which may partially con-tribute to its anti-angiogenesis effect. Although we did not detect other molecular and cellular pathways by which marchantin C causes regres-sion of CAM vessels, this result certainly suggests that it could be a reasonable hypothesis and deserves investigations in the future studies.

In conclusion, we demonstrate that marchantin C influences the migration and invasion of brain can-cer cells and is capable of inhibiting angiogenesis at low concentrations. Marchantin C appears to affect MMP-2 activity via the MAPK pathway. It also has anti-angiogenic effects. Further study is necessary to elucidate the complete mechanism for marchantin C induction of brain cancer invasion.

Materials and Methods

Chemicals and antibodies. Rabbit anti-phospho-ERK1/2 antibody, anti-ERK1/2 antibody, anti-phospho-Stat3 (Ser 727) antibody, anti-Stat3 antibody, anti-phospho-AKT (Ser 473) antibody, anti-AKT antibody, anti-β-actin antibody and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (New England BioLabs, USA). Rabbit anti-MMP-2 antibody was purchased from Santa Cruz Biotechnology, Inc., USA. The structure of marchantin C, isolated from Dumortiera angust was identified by interpretation of spectral data (MS, 1H NMR, 13C NMR, 2D NMR) as described previously.8 ten mM marchantin C dis-solved in dimethylsulfoxide (DMSO) was stored at -20°C as a stock solution and suitable dilutions were used according to experimental requirements.

Cell culture. Human malignant glioma cell lines, T98G and U87 cells were purchased from the cell bank of the Chinese

Figure 4. Marchantin C decreased the protein expression of p-eRK1/2. Marchantin C treatment decreased the phosphorylation of eRK1/2 of T98G (a) and U87 (B) cells in a dose-dependent manner by using western blot, while total sTaT3, aKT and eRK1/2 levels and phosphorylation of sTaT3 and aKT were not affected. (C) The histogram represented the ratio of p-eRK1/2 expression of each treatment group to the control group (*p < 0.01).


inverted microscope (Olympus, Japan) at the indicated time points from exactly the same position (x10 objective). The gap width of the zero time point scratch was set at 100%, measured by Image-Pro Plus software, and the mean percentage of the total size of the gap areas were calculated at different time points.

Boyden chamber assay. The modified Boyden chamber assay used for analysis of cell migration is based on a chamber with two medium filled compartments. Migration assays were carried out in a 24-well chemotaxis chamber (Neuro-Probe, Gaithersburg, Maryland) with 8.0 μm pore polycarbonate filter inserts. The insert was coated overnight with 0.1% w/v gelatine and air-dried.25 T98G and U87 glioma cells, suspended in serum-free medium at 1 x 105/250 μl, were treated with marchantin C at different concentrations (0, 6, 8 and 10 μM) in the upper cham-ber. Medium containing 10% fetal calf serum was added to the lower chamber. Cells were allowed to migrate for 24 h at 37°C in a humidified atmosphere containing 5% CO

2. The upper side

of the membrane was wiped by cotton swabs. Cells on the lower side of the membrane were stained with 0.1% crystal violet and counted using a light microscope (x20, objective). Six random fields per filter were counted and data were presented as the num-ber of cells penetrating through the membrane.

Cell invasion assay. Cell invasion assays were carried out using 24-well Transwell chamber with 8.0 μm pore polycar-bonate filter inserts (Costar, USA) that had been coated with 50 μl of Matrigel (BD Biosciences, USA; 1:10 dilution in serum-free medium) as described previously.25 1 x 105 T98G and U87 glioma cells in serum-free medium were seeded to the top of the chamber and the bottom chamber was filled with medium con-taining 10% serum in the absence (control) or presence of indi-cated agents. After 24 h of incubation in a 5% CO

2 humidified

chamber at 37°C, noninvading cells were removed by wiping the upper surface of the membrane with a cotton swab and cells that migrated through the Matrigel were stained with 0.1% crystal violet. Six random fields per filter were counted under a light microscope (x20, objective) and each experiment was performed in triplicate.

Western blot analysis. As described previously,9 cell samples were lysed in 20 mM Tris-HCl buffer (pH 7.4), 150 mM NaCl, 1 mM Na

3VO

4, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF,

50 mM NaF and 1% NP-40. Whole cell extracts (30 μg/lane) were separated by 10% SDS-PAGE. After electrophoresis, pro-teins were transferred to PVDF membranes (Millipore, Bedford, MA) and blocked with 5% non-fat milk for at least 1 h fol-lowed by an overnight blotting with primary antibody at 4°C. Membranes were washed with TBST three times before being incubated with secondary antibody conjugated with horseradish peroxidase, then washed three times with TBST. Proteins were detected by the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL) using X-ray film. The bands were exam-ined using densitometry with AlphaEaseFC software (Version 4.0.0, Alpha Innotech Corp.).

Chick chorioallantoic membrane assay. CAM assay was performed to evaluate the ability of marchantin C to inhibit angiogenesis in vivo. Briefly, fertilized chicken eggs were incu-bated at 37.5°C with 70% humidity for 7 d. Eggs were candled

a density of 2 x 104 cells/well in 48-well plates (Costar). After 24 h, the cells were treated with a range of doses of marchantin C. The number of viable cells (unstained) versus dead cells (blue stained) was calculated by using a hemacytometer. About 90% viability was regarded as no toxicity.

Scratch-induced migration assay. To study directional cell migration, scratch assays were performed.24 Briefly, U87 and T98G cells were seeded on collagen-covered 12-well plates at a density of 2 x 105 cells/well. Confluent monolayers were washed with PBS and three artificial gaps were created by scraping with a pipette tip in each well. The cells were washed twice with PBS before their subsequent incubation with culture medium in the absence (control) or presence of marchantin C at appropriate concentrations. Gap closure was monitored and photographs of treated cells moving within the scratch were taken with an

Figure 5. Inhibition of in vivo angiogenesis by marchantin C in chick embryo chorioallantoic membrane (CaM) assay. Gelatin sponges (desig-nated by circles) containing 20 μl PBs alone or 20 μl PBs with marchan-tin C at concentrations of 6, 8 and 10 μM were placed on chick embryo chorioallantoic membranes and incubated for 48 h. Photograph were taken and macroscopic assessment of vascular density conducted by counting the number of blood vessels within an area surrounding the gelatin sponges. The relative inhibition of angiogenesis was 23.7, 32.8 and 55.9% in the presence of 6, 8 and 10 μM of marchantin C, respec-tively, as compared to the control (*p < 0.01).


zone around the gelatin sponge as described.26 At least three CAMs were used for each experiment.

Statistical analysis. Data was expressed as mean ± SD from at least three independent experiments. Statistical analysis was per-formed with Student’s t-test. p < 0.05 was considered statistically significant in all cases.

Acknowledgements

This work was supported by Natural Science Foundation of China (#30571696; #30671901; #30628014) and Ministry of Science and Technology of China (#2007AA021000; #2004CB518807).

to confirm embryo growth and to locate vascularized areas of the CAM. A window was then opened on the eggshell in order to expose the CAM. The window was covered with sterile parafilm and the eggs were placed back into the incubator. After 24 h incu-bation, a piece of gelatine sponge (5 mm in diameter) containing 20 μL of PBS alone as a control or 20 μL PBS with marchantin C at concentrations of 6, 8 and 10 μM were applied to the mem-brane under sterile conditions. After 48 h incubation at 37.5°C, photographs were taken and angiogenesis was assessed as the number of visible blood vessel branch points within a defined

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Marchantin C a Potential Anti-Invasion Agent in Glioma Cells

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