JSI-124 Suppresses Invasion and Angiogenesis of Glioblastoma Cells In Vitro

Glioblastoma multiforme (GBM) is one of the utmost malignant tumors. Excessive angiogenesis and invasiveness are the major reasons for their uncontrolled growth and resistance toward conventional strategies resulting in poor prognosis. In this study, we found that low-dose JSI-124 reduced invasiveness and tumorigenicity of GBM cells. JSI-124 effectively inhibited VEGF expression in GBM cells. In a coculture study, JSI-124 completely prevented U87MG cell–mediated capillary formation of HUVECs and the migration of HUVECs when cultured alone or cocultured with U87MG cells. Furthermore, JSI-124 inhibited VEGF-induced cell proliferation, motility, invasion and the formation of capillary-like structures in HUVECs in a dose-dependent manner. JSI-124 suppressed VEGF-induced p-VEGFR2 activity through STAT3 signaling cascade in HUVECs. Immunohistochemistry analysis showed that the expression of CD34, Ki67, p-STAT3 and p-VEGFR2 protein in xenografts was remarkably decreased. Taken together, our findings provide the first evidence that JSI-124 effectively inhibits tumor angiogenesis and invasion, which might be a viable drug in anti-angiogenesis and anti-invasion therapies.


Introduction
Glioblastoma multiforme (GBM), the most aggressive and accounts for 54% of all gliomas [1], is considered incurable largely due to sustained and excessive angiogenesis and invasiveness, and approximately 77% of glioma patients die within the first year of their diagnosis [2][3][4]. Angiogenesis, considered crucial for the transition of tumors from a dormant to malignant state [5,6], is now established as one of the hallmarks of cancer and responsible for over 90% of all cancer deaths [7]. Angiogenesis is a rate-limiting process including the destabilization of integrated blood vessel, endothelial cell proliferation, migration, and tubulogenesis. Disrupting tumor angiogenesis has been shown effective tumor growth and metastasis inhibition [8].

Cell viability assay
The cytotoxic effect of JSI-124 on GBM cells and HUVECs were determined using CCK-8 assay (Dojindo, Japan). Cells in medium containing 20% FBS or 10% FBS were seeded into 96well flat-bottomed plates at 5×10 3 cells/well and incubated at 37°C overnight. After the desired treatment, the cells were incubated for an additional 4 h with 100μl serum free DMEM with 10μl CCK-8 at 37°C. The absorbance at 450 nm was measured using a microplate reader. The absorbance was measured at 450 nm wavelength.

Western blot analysis
After the desired treatment, cells were washed twice with cold phosphate-buffered saline (PBS) and harvested with a rubber scraper. Cell pellets were lysed and kept on ice for at least 30 min in a buffer containing 50 mM TrisHCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride and 1mM PMSF. The lysates were cleared by centrifugation and the supernatants were collected. Cell lysates were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to western blot analysis with the primary antibodies and horseradish peroxidase-labeled secondary antibodies.

VEGF Enzyme-Linked Immunosorbent Assay
The VEGF protein that released into the conditioned medium of U87MG cells was determined using an ELISA kit (R&D Systems, Minneapolis, MN, USA). U87MG cells (5×10 5 ) were seeded in six-well plates in 2ml of complete growth medium. Twenty-four hours later, cells were serum-starved for 24 hours and then exposed to JSI-124 (100 nM) with 1 ml of DMEM containing 2% FBS. After 24 hours of incubation in 5% CO2 at 37°C and 95% humidified air to allow VEGF protein secretion, the conditioned medium was collected, and 1 mM phenyl methyl sulphonyl fluoride (PMSF) was added. The supernatant was clarified by centrifugation for 5 minutes at 14,000 rpm, aliquoted, and stored at -80°C until analysis.
Clonogenicity assay U251 and U87MG cells were pretreated with DMSO (<0.1%) or JSI-124 (100 nM) for 2 h. The pretreated cells were throughoutly washed with serum-free medium for three times to remove all drugs. U251 (1×10 3 ) and U87MG (1×10 3 ) cells then were plated onto a 6-well tissue culture plate in complete medium and incubated at 37°C. Cells were allowed to grow in complete medium for 5 days. Then cells were fixed and stained with 1% Toluidine Blue in 1% borax and counted under the microscope (×50 magnification). Five random fields were counted under a light microscope at ×50 magnification.

Endothelial cell capillary-like tube formation assay
Matrigel was thawed at 4°C, and each well of prechilled 96-well plates was coated with 30 μl matrigel and incubated at 37°C for 45 min. HUVECs (4×10 4 ) were added in 200 μl ECM with various concentrations of JSI-124. After 4 h of incubation at 37°C, 5% CO2, tubular structure formation was captured under microscope and measured length of tube by using Image-Pro Plus software.
To examine the effect of JSI-124 on tumor cell-induced tube formation of HUVECs, a conditioned medium was collected from U87MG cells and used as the growth medium for HUVECs. Briefly, cells were seeded at 70% confluency; after overnight incubation, cells were treated in the presence or in the absence of JSI-124, as indicated, for 8 hours. After 8 hours, cells were washed thoroughly with phosphate-buffered saline (PBS) and further incubated in reduced serum containing DMEM for another 24 hours and collected as a conditioned medium. The conditioned medium was then used to study the in vitro tube formation assay in HUVECs, as described above.
To examine the effect of JSI-124 on VEGF-induced tube formation, HUVECs suspended in endothelial cell basal medium containing 0.5% fetal bovine serum (FBS) were seeded on a culture plate coated with growth factor-reduced Matrigel. JSI-124 was added to the cell suspension 30 minutes before plating the cells, and recombinant human VEGF 165 (20 ng/ml) was added at the time of seeding as indicated.

Transwell migration and invasion assay
The motility and invasion of HUVECs and GBM cells were determined using a transwell assay (Corning, Inc.) with 6.5-mm-diameter polycarbonate filters (8-μm pore size). The chambers of transwell invasion assay were coated 50% matrigel, while transwell migration assay coated no matrigel. The two chambers were coated with 0.1% gelatin for 30 min in cell incubator. The bottom transwell chambers were filled with ECM or DMEM with 0.5% FBS supplemented with or without 20ng/mL VEGF, and the top chambers were seeded 4×10 4 cells/per well HUVECs or GBM cells in 100 μL ECM or DMEM (0.5% FBS) plus different concentrations of JSI-124. Cells were allowed to migrate for 8 h. To assay for glioblastoma cell-induced migration of endothelial cells, we performed a coculture assay using migration chambers as described by Tsujii et al [23]. Nonmigrated or noninvasive cells were removed with cotton swabs, and migrated or invasive cells were fixed with cold 4% paraformaldehyde and stained with eosine or crystal violet. Images were taken using an inverted microscope.

Cell death detection ELISA Plus assay
Cell death detection ELISA Plus assay (Roche) was performed to determine apoptosis by quantification of histone-complexed DNA fragments according to the manufacturer's instruction and absorbance was determined at 405 nm wavelength.

Flow cytometry assay
After treating cells with various treatments, we measured apoptosis using Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen, Ltd, USA) according to the manufacturer's instructions. Briefly, cells were washed twice with cold PBS and collected and then resuspended in Annexin V binding buffer at a concentration of 5× 10 6 cells/ml. Afterward, 1×10 6 cells were transferred to a tube and subsequently stained with 5μl Alexa Fluor 488 annexin V and 100μg/ ml propidium iodide (PI) to each 100μl of cell suspension. After incubated in the dark for 15 min at room temperature, stained cells were then analyzed by flow cytometry.

Tumor xenograft model
Balb/c nude (nu/nu) female mice were purchased from Vital River Laboratories. U87MG cells (5×10 6 cells in 50μl of serum-free DMEM) were inoculated subcutaneously into the right flank of five-week-old female mice after acclimated for a week. Tumor growth was measured daily with calipers. Tumor volume was calculated as (L×W 2 )/2, where L is the length in millimeters, and W is the width in millimeters. When the tumors reached a mean volume of 90 to 120 mm 3 , 12 mice were randomly assigned to JSI-124 (1 mg/kg/day, in 20% DMSO in PBS) or drug vehicle control (20% DMSO in PBS) and dosed i.p. with 100μl vehicle of drug once daily for 18 days. Tumors were dissected and frozen in liquid nitrogen or fixed in formalin.

Statistical analysis
The data were expressed as means ±S.D. Statistical analysis was performed with the two-tailed Student's test. The criterion for statistical significance was set at P<0.05.

JSI-124 inhibits cell viability and induces apoptosis in GBM cells
To assess the effect of JSI-124 on GBM cells, we first examined several GBM cell lines treated with various doses of JSI-124 by CCK-8 assay. JSI-124 inhibited cell viability in a dose-and time-dependent manner, and significant cell viability inhibitory effect of JSI-124 was observed at concentrations more than 100nmol/L (Fig. 1A). JSI-124 exhibited anticancer potential through the induction of apoptosis in a wide variety of human tumor cell lines [19,21,22]. To verify apoptotic property on GBM cells, flow cytometry assay and cell death detection ELISA-Plus assay were performed. No change in apoptosis was observed at 100nM JSI-124 for 48 h, but higher concentration JSI-124 significantly induced GBM cells apoptosis dose-dependently ( Fig. 1B and S1 Fig.). The similar phenomenon was observed in GBM cells determined by cell death detection ELISA Plus assay (Fig. 1C).

Exposure of GBM cells to JSI-124 at low dose reduces clonogenicity
We further examined if low-dose JSI-124 would be sufficient to prevent GBM cells tumorigenicity. Using an in vitro clonogenic assay, we found that just brief exposure of GBM cells to JSI-124 (100 nM) for 2 h was sufficient to significantly reduce the number of tumor colonies of U251 and U87MG cells (Fig. 3), suggesting that JSI-124 may inhibit GBM tumorigenicity.

JSI-124 inhibits VEGF expression in GBM cells
VEGF is a critical factor in new blood vessel formation [6,24]. In a tumor microenvironment, cancer cells secrete a high level of VEGF that binds to receptors on surrounding endothelial cells, promoting endothelial cell migration, proliferation, and differentiation, as well as tube formation [25,26]. In this experiment, we measure the effect of JSI-124 on the VEGF level in GBM cells at different concentrations by ELISA. The treatment of JSI-124 at 100nM for 24 hours markedly reduced the secretion of VEGF by GBM cells (Fig. 4).

JSI-124 inhibites the U87MG cells-induced tube formation of HUVECs
In this experiment, we used conditioned media from U87MG cells treated with or without JSI-124 to determine whether GBM cells could induce the capillary formation of HUVECs and examine the effect of on this event. It is clearly seen that the conditioned medium (without JSI-124) from U87MG cells induced capillary formation of HUVECs. However, the conditioned medium (with JSI-124) from U87MG cells produced a complete prevention of tube formation (Fig. 5A). We also measured the VEGF level in the conditioned medium obtained from U87MG cells, a marked inhibition of VEGF production was noticed in the presence of JSI-124 (Fig. 5B). These results demonstrated that the capillary formation of HUVECs was augmented by GBM cells and that this activity was downregulated after JSI-124 treatment.

JSI-124 inhibites the U87MG cells-induced migration of HUVECs
We further examined the effect of JSI-124 on HUVECs migration induced by GBM cells using a coculture assay. The HUVECs cocultured with U87MG cells (Fig. 6B) triggered pronounced migration compared with HUVECs alone (Fig. 6A), while this increased migration of HUVECs was prevented significantly when U87MG cells were treated with JSI-124 (Fig. 6C). These results revealed an increased migration of endothelial cells when cocultured with GBM cells, and this migration was severely inhibited when GBM cells were treated with JSI-124.

JSI-124 inhibits cell viability and induces apoptosis in HUVECs
To systematically address the inhibitory activity of JSI-124 on HUVECs growth, we first evaluated the cell viability by CCK-8 assay. Treatment with JSI-124 resulted in growth inhibition in HUVECs in a dose-dependent manner (Fig. 7A). We then examined the apoptotic effects of JSI-124 by flow cytometry and cell death detection ELISA Plus assay. We found that the proportion of apoptotic cells was significantly increased in a dose-dependent manner in JSI-124-treated HUVECs for 48h ( Fig. 7B and S2 Fig.). Similar results were found in HUVECs performed by cell death detection ELISA Plus assay (Fig. 7C). Moreover, JSI-124 treatment for 48h down-regulated the expression of p-STAT3 (Ser727) (Fig. 7D). In addition, Bcl-2 and Bcl-xL significantly decreased as well as clear cleavages of PARP and caspase-3 occurred dose-dependently (Fig. 7E).

JSI-124 inhibits VEGF-induced migration, invasion and tubular structure formation of HUVECs
To assess the antiangiogenic effect of JSI-124 on endothelial cells, cell viability of HUVECs was determined by CCK-8 assay. As shown in Fig. 8A, the proliferation of HUVECs stimulated by VEGF (20ng/ml) was markedly decreased after JSI-124 treatment ranging from 25 to 100nmol/ L for 24 h indicating extracellular VEGF acted as a strong stimulus for HUVECs proliferation. Cell migration is an essential step for endothelial cell to form blood vessels in angiogenesis [27]. Next transwell migration assay and transwell invasion assay were utilized to investigate the inhibitory effects of JSI-124 on the motility of HUVECs. The results showed that JSI-124 inhibited VEGF-induced HUVEC migration (Fig. 8B) and invasion (Fig. 8C) in a dosedependent manner. The maturation of migrated endothelial cells into a capillary tube is a critical step during angiogenesis [28]. To examine the potential effects of JSI-124 on the tubular structure formation, we conducted two-dimensional matrigel assays and examined JSI-124 effect on tubular structure formation in HUVECs. VEGF significantly enhanced the tubular network formation compared to HUVECs seeded on matrigel under 0.5% serum in ECM alone; however, treatment with JSI-124 strongly inhibited the VEGF-stimulated tubular network formation, and tube length measurement showed that JSI-124 treatment inhibited the tube length dose-dependently   (Fig. 8D). Overall, these results indicated that JSI-124 could suppress VEGF-induced angiogenesis by inhibiting migration, invasion, and tube formation of HUVECs.

JSI-124 inhibits activation of VEGFR2 and JAK2/STAT3 signaling induced by VEGF in HUVECs
VEGFR2 activation is responsible for endothelial cell migration and proliferation [29]. We examined the effects of JSI-124 on phosphorylation of VEGFR2 to determine its inhibitory effect on VEGFR2-mediated signaling pathways in HUVECs. We found that VEGFR2 was strongly phosphorylated by exogenous VEGF to HUVECs, while treatment with JSI-124 significantly blocked VEGF-induced phosphorylation of VEGFR2 in a dose-dependent manner without affecting overall VEGFR2 expression levels (Fig. 9A). Previous studies suggested that VEGF triggered the activation of STAT3 signaling in HUVECs [30][31][32]. The effects of JSI-124 on the JAK2/STAT3 signaling were determined by using western blot analysis. In addition, JSI-124 with VEGF. After about 8 h, the migrated HUVECs passed through the membrane were quantified. (D) JSI-124 inhibits VEGF-induced tube formation of HUVECs determined by capillary-like tube formation assay. Cells (4×10 4 / well) were placed in the 96-well plates coated with matrigel. After 4 h of incubation, tubular structure formation was captured under microscope. All data were expressed as mean ± SD. * P < 0.05; ** P < 0.01 versus VEGF alone.

JSI-124 inhibits tumor growth and angiogenesis in U87MG cells xenograft model
To evaluate the effects of JSI-124 on tumor growth and tumor angiogenesis in vivo, we further constructed a series of therapeutic experiments using U87MG cells xenograft mouse model. No major side effects were noted throughout the study. We found that intraperitoneal administration of JSI-124 (1mg/kg/d, 18 days) markedly inhibited tumor volumn and tumor weight as compared with the counterparts treated with DMSO. The average tumor volume of solid tumors in JSI-124-treated mice was 432 mm 3 (±90), as compared with 1210 mm 3 (±230) for control group (Fig. 10A). Moreover, there was no effect on body weight of mice (Fig. 10B). The average tumor weights at study termination were 1,450 mg (±285) and 446 mg (±96) in control and JSI-124 group, respectively (Fig. 10C).
We further showed the immunohistochemical staining results of Ki67, CD34, p-VEGFR2 and p-STAT3, on tumor sections. As shown in Fig. 10D, intraperitoneal injections of JSI-124 resulted in pronounced decrease in tumor cell proliferation and CD34-positive endothelial cells, and marked reduction expression of p-VEGFR2 and p-STAT3 in solid tumors.

Discussion
Glioblastoma, the most common brain tumor and are associated with high invasion, is resistant to conventional treatment methods and have poor prognosis mainly due to uncontrolled angiogenesis and resultant high tumor mass [9]. The current radioactive and chemotherapeutic regimens also kill non-specifically functional and non-cancerous cells in the brain, which might adversely impair different functions of the body. To prevent this highly invasive cancer from occurring, preventive approaches are highly desirable. In this study, we demonstrated for the first time that JSI-124 reduced invasiveness and tumorigenicity of GBM cells at low dose. JSI-124 effectively inhibited VEGF expression in GBM cells. In addition, JSI-124 prevented U87MG cell-mediated capillary formation of HUVECs and the migration of HUVECs when cultured alone or cocultured with U87MG cells. Moreover, JSI-124 significantly inhibited angiogenesis including human endothelial cell proliferation, migration, invasion and capillary structure formation in a concentration-dependent manner as well as the stimulative effects of human endothelial cell death in response to VEGF in vitro. Immunohistochemistry analysis showed that the expression of CD34, Ki67, p-STAT3 and p-VEGFR2 protein in xenografts was remarkably decreased. Taken together, our results suggested that JSI-124 may be a potent chemopreventive agent for GBM with anti-invasion and anti-angiogenisitic activities.
JSI-124 has been reported to inhibit the proliferative activity of several types of cancer cells associated with apoptotic cell death mediated via the inhibition of the constitutively activated STAT3 signaling pathway [19,21,33,34]. Consistent with these findings, our results showed that JSI-124 induced apoptosis dose-dependently in GBM at higher than 100 nM. GBMs are highly infiltrative into brain preventing surgical cure even with heroic resections [35,36], invading tumor cells appear to be particularly resistant to cytotoxic therapy and are often protected by an intact neurovascular unit [37]. Our data provided the evidence that invasiveness of GBM cells was significantly inhibited after low-dose JSI-124 treatment Moreover, we showed that brief exposure to low-dose JSI-124 can reduce the clonogenicity of GBM cells in vitro.
The importance of tumor angiogenesis in cancer progression is underscored by the fact that it is an important target for the development of anticancer therapies based on the inhibition of angiogenesis, and antiangiogenic therapy is now considered as the forth strategy to treat cancer [8,38,39]. Angiogenesis is a complex multistep process that involves endothelial cell proliferation, migration, and tube formation triggered by specific growth factors in tumor microenvironment [39]. Cancer cells produce numerous angiogenic factors, including VEGF, FGF, EGF, PDGF etc, which play a pivotal role in the development of tumor angiogenesis by stimulating endothelial cell proliferation, migration, and capillary tube formation [40][41][42]. Among all angiogenic factors, VEGF is identified as a key mediator of angiogenesis [6]. Glioblastomas secrete a very large quantity of VEGF protein into the surrounding microenvironment, thereby allowing endothelial cell proliferation, migration, and tube formation [43,44]. In this study, we found that the basal VEGF levels in U87MG and U251 cells were significantly high and that JSI-124 dose-dependently downregulated protein levels of VEGF. Using a coculture assay, we examined the effect of this inhibitor on glioblastoma cell-induced migration and on the tube formation of endothelial cells. Treatment with JSI-124 decreased VEGF secretion by U87MG cells and thereby reduced endothelial cell migration and tube formation.
Moreover, we showed that JSI-124 blocked the proliferation, capillary formation, and migration of endothelial cells, all of which are critical steps for angiogenesis. We further showed that induction of apoptosis in endothelial cells by JSI-124 was due to the downregulation of Bcl-2 and Bcl-xL, both of which are apoptotic proteins overexpressed in endothelial cells and associated with resistance to apoptosis. Overexpression of VEGF and VEGF receptors correlates with increased microvessel density, proliferation, and tumor growth rate, which lead to poor patient prognosis in a variety of malignancies [45,46]. We provided first evidence that JSI-124 effectively abrogated VEGF-induced HUVECs proliferation, invasion, migration, and capillary-like structures formation in vitro. Phosphorylation of VEGFR2 is critical for VEGFmediated microvascular permeability, endothelial cell proliferation, invasion and migration [47][48][49]. In the present study, we found that JSI-124 blocked the activity of VEGFR2 (Tyr 1175 ) by down-regulation of VEGF-induced phosphorylation of VEGFR-2 expression. A close association between STAT3 activation and glioma growth and vascularization has been reported previously [11,12,50], and activation of STAT3 has been directly correlated with VEGF production [13]. Our present investigation showed that the expression of VEGF was dose-dependently suppressed by JSI-124 via STAT3 (Ser 727 ) inhibition in HUVECs. STAT3 is principally activated by nonreceptor tyrosine kinase JAK2 [51]. Our results showed that phosphorylation of JAK2 (Tyr 1007/1008 ) was dose-dependently blocked by JSI-124 in HUVECs, indicating that the direct effects of JSI-124 on angiogenesis might be through inhibiting the VEGFR2/STAT3 signaling pathway.
To evaluate the antitumor activity of JSI-124 in vivo, Babl/c nude mice transplanted with U87MG cells were treated with JSI-124. We found that JSI-124 (1 mg/kg/day) significantly suppressed tumor volume and tumor weight without any side effects on mice, and remarkably reduced neovascularization accompanied by down-regulation expression of p-STAT3 and p-VEGFR2. In addition to the effective antiangiogenesis of JSI-124 in vitro, we presume that JSI-124 suppresses GBM growth in vivo through not only directly tumor cell proliferation inhibition but also tumor angiogenesis suppression.
Taken together, our results indicate that JSI-24 may be an effective preventive agent for GBM with anti-invasion and anti-angiogenesitic activity. It is hoped that in the future, patients harboring GBM can be offered an affordable preventive agent to reduce the incidence of GBM.