DYT-40, a novel synthetic 2-styryl-5-nitroimidazole derivative, blocks malignant glioblastoma growth and invasion by inhibiting AEG-1 and NF-κB signaling pathways

Astrocyte elevated gene-1 (AEG-1) has been explored as a novel target for human glioma therapy, thus reflecting its potential contribution to gliomagenesis. In the present study, we investigated the effect of DYT-40, a novel synthetic 2-styryl-5-nitroimidazole derivative, on cell growth and invasion in glioblastoma (GBM) and uncovered the underlying mechanisms of this molecule. DYT-40 induces the intrinsic mitochondrial pathway of apoptosis and inhibits the epithelial-mesenchymal transition (EMT) and invasion of GBM cell lines. Furthermore, DYT-40 deactivates PI3K/Akt and MAPK pathways, suppresses AEG-1 expression, and inhibits NF-κB nuclear translocation. DYT-40 reduced the tumor volumes in a rat C6 glioma model by apoptotic induction. Moreover, HE staining demonstrated that the glioma rat model treated with DYT-40 exhibited better defined tumor margins and fewer invasive cells to the contralateral striatum compared with the vehicle control and temozolomide-treated rats. Microscopic examination showed a decrease in AEG-1-positive cells in DYT-40-treated rats compared with the untreated controls. DYT-40-treatment increases the in vivo apoptotic response of glioma cells to DYT-40 treatment by TUNEL staining. In conclusion, the inhibitory effects of DYT-40 on growth and invasion in GBM suggest that DYT-40 might be a potential AEG-1 inhibitor to prevent the growth and motility of malignant glioma.

neuroblastoma, and hepatocellular carcinoma cells [8][9][10][11] . In contrast, the knockdown of AEG-1 expression significantly inhibits these phenotypes in malignant glioma and neuroblastoma 11,12 . Previous studies have demonstrated that the ectopic over-expression of AEG-1 promoted epithelial-mesenchymal transition (EMT), which resulted from the down-regulation of E-cadherin and the up-regulation of vimentin in lung cancer cell lines and clinical lung cancer specimens 13 . In these contexts, AEG-1 might provide a viable target for clinical therapeutic intervention in the EMT-mediated invasion of carcinomas.
Ras activation initiates a complex axis of transduction, including the Raf/MAPK (ERK) pathway, originally involved in the plasma membrane-to-nucleus signaling crucial for cell mitogen-mediated proliferation 14 and the phosphatidylinositol 3-kinase (PI3K) Akt pathway, which is involved in cell survival signaling 15 . Akt stabilizes C-myc via phosphorylation and inhibits the activation of GSK-3β , which promotes the transcriptional activation of C-myc [16][17][18][19] . The mammalian NF-κ B family includes p50 (NF-κ B1), p52 (NF-κ B2), p65 (ReLA, NF-κ B3), ReL and ReLB, which share the amino-terminal ReL homology domain RHD and are regulated by the eight Iκ B family members 20 . Previous studies have shown that AEG-1 is an important positive regulator of nuclear factor kappa-B p65 (NF-κ B) and that the activation of NF-κ B p65, which is induced by AEG-1, exhibits a key molecular mechanism in which AEG-1 promotes cell growth and invasion in malignant glioma cells 8,21 .
DYT-40 (referred to as compound 3c in a previous study) is a novel 2-styryl-5-nitroimidazole derivative containing the 1,4-benzodioxan moiety (3a-3r). These compounds (3a-3r) have been synthesized, biologically evaluated, and demonstrated to be FAK inhibitors in molecular docking studies 22 . Among all compounds, 3p exhibits significant FAK inhibitory activity (IC 50 = 0.45 μ M) and possesses good A549 anti-proliferative activity. However, the FAK inhibitory effect of compound 3c (DYT-40, IC 50 = 18.42 μ M) is not as good as that of compound 3p. Although 3p showed the most potent activity in vitro which inhibited the growth of adenocarcinomic human alveolar basal epithelial cells A549 with IC 50 value of 3.11 μ M and human cervical cancer cells Hela with IC 50 value of 2.54 μ M respectively, the efficacy of DYT-40 on glioma cells growth seems to be better than 3p.
The present study provides the first evidence that DYT-40 represses the expression of AEG-1 and the activation of the NF-κ B pathway, which plays an important role in tumor development and progression 6,8 .

Reagents and antibodies.
Cell proliferation assay. Cell growth was analyzed using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Japan) according to the manufacturer's instructions 24 . Briefly, the cells were seeded at a density of 5 × 10 3 cells/well onto 96-well plates and treated with various concentrations of DYT-40 (0.005, 0.05, 0.5, 5 and 50 μ M) and cultured for 24, 48 and 72 h at 37 °C in 5% CO 2 . Subsequently, the medium in each well was substituted with 100 μ L of fresh medium containing 10% Cell Counting   average absorbance of control group ×100. The IC 50 was defined as the concentration of DYT-40 that reduced cell viability by 50%, calculated using the logit method. All assays were performed in triplicate.
Annexin V/PI Staining. The Annexin V/PI Staining Apoptosis-mediated cell death of U251 and U87 cells was examined using the Annexin V-FITC Apoptosis Detection Kit I (Catalog Number: 556547, BD Biosciences, Qume Drive, San Jose, CA, USA) according to the manufacturer's instructions. Briefly, 1 × 10 6 cells were harvested and washed with PBS. Subsequently, the cells were re-suspended in 500 μ L of binding buffer, and 5 μ L Annexin-V-FITC and 1 μ L PI were added, followed by incubation for 10 min in the dark at room temperature 23,25 .
The cells were analyzed using the BD FACSCalibur ™ Flow Cytometry System (BD Biosciences, Franklin Lakes, NJ) and the Cell Quest software (BD Biosciences). Cells in the early stages of apoptosis were Annexin V positive and PI negative, whereas cells in the late stages of apoptosis were both Annexin V and PI positive. DAPI staining assay. To observe the morphology of apoptosis, the cell nuclei were visualized following DNA staining. Briefly, the cells were seeded onto six-well tissue culture plates at a concentration of 1 × 10 5 cells/well and treated with the indicated concentrations of DYT-40 (0, 5, 10 and 20 μ M). In addition, cells pretreated or not with pcDNA3.1-AEG-1 (1 μ g) were incubated with 20 μ M DYT-40 and subsequently fixed with 4% paraformaldehyde for 20 min and washed with PBS, followed by incubation with the fluorochrome dye 4′ , 6-diamidino-2-phenylindole (DAPI, 1 μ g/mL, Sigma-Aldrich) for 10 min. After washing with PBS, the cells were observed using fluorescence microscopy (Olympus, Tokyo, Japan) with a peak excitation wavelength of 340 nm 25 . Cell adhesion assay. The cell adhesion assay was performed as previously described 26 , with some modifications. The 96-well plates were coated with fibronectin (5 μ g/mL) at 4 °C overnight and subsequently blocked in BSA (1%) for 1 h. Serum-starved cells were exposed to DYT-40 (2.5, 5, or 10 μ M) for 24 h prior to seeding. The target cells were harvested and suspended in serum-free medium. The cells (2 × 10 5 /mL) were seeded onto fibronectin-coated plates and subsequently incubated at 37 °C for 1 h. Non-adherent cells were removed after gentle washing with PBS. Subsequently, the colorimetric MTT assay was employed to analyze the cell adhesion.
Transwell invasion assay. The transwell migration assay was performed using 24-well MILLI Cell Hanging Cell Culture inserts 8 mm PET (Millipore, Bedford, MA, USA) as previously described 27 . The cells were cultured in DYT-40 (2.5, 5 and 10 μ M) for 24 h, and in parallel, pcDNA3.1-AEG-1 was transfected into U251 and U87 cells for 24 h and treated with DYT-40 (10 μ M) for an additional 24 h. Subsequently, the cells were seeded in serum-free medium onto triplicate wells of BD BioCoat TM Matrigel TM Invasion Chambers (BD Bioscience), and complete medium containing 10% fetal bovine serum was added to the lower chamber. The cells invaded through the polycarbonate membrane were stained using crystal violet after incubation for 24 h. Image magnification: 200× . Five random fields from each of the triplicate invasion assays were counted using phase-contrast microscopy. Transient transfection. The pcDNA3.1-AEG-1/MTDH plasmid was kindly provided from Dr.
Quantitative real-time polymerase chain reaction. Total RNA was isolated using TRIzol reagent, according to the manufacturer's instructions. First-strand cDNA was synthesized using total RNA (1 μ g) at 70 °C for 5 min, 42 °C for 60 min, and 95 °C for 10 min using an oligo (dT) 12-18 primer and subjected to real-time PCR. Amplification assays were performed on a 7900 Fast Real-Time PCR System using the SDS software (Applied Biosystems) in 10-μ L reactions containing 0.2 μ M of each primer, 5 μ L SYBR Green PCR master mix (2× ) and 0.2 μ L cDNA. After an initial denaturation at 95 °C for 30 s, amplifications were performed for 40 cycles at 95 °C for 5 s and 60 °C for 31 s. The signals from each target gene were normalized based on the corresponding GAPDH signal. The following PCR primers were used in the present study: (1) AEG-1 F: CTAGTATCCTGGTTTAACAACAGTGCCCTGTTTACAACAGATTGTGCCCTATCTCATCA, AEG-1 R: AGCTTGATGAGATAGGGCACAATCTGTTGTAAACAGGGCACTGTTGTTAAACCAGGATA; (2) GAPDH F: CAGTCCATGCCATCACTGCCA, GAPDH R: CAGTGTAGCCCAGGATGCCCTT.
Western blotting analysis. Briefly, after washing twice with PBS, the cultured cells were collected and lysed in lysis buffer (100 mM Tris-HCl, pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 200 mM β -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/mL aprotinin). Nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (KeyGen, Nanjing, China) according to the manufacturer's instructions. The lysates were centrifuged at 13,000 × g for 15 min at 4 °C. The concentration of total proteins was measured using the BCA assay method with a Varioskan spectrofluorometer and spectrophotometer (Thermo, Waltham, MA) at 562 nm 25 . The protein samples were separated on a 12% SDS-PAGE gel and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA). The immune complexes were formed after the incubation of the proteins with primary antibodies overnight at 4 °C. After incubation with the appropriate secondary antibodies, the blots were visualized using ECL plus western blotting detection reagents (Bio-Rad) and a ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA). The densitometric analysis of the band intensity was performed using Image lab software (Bio-Rad, Hercules, CA, USA).
Immunofluorescence and confocal fluorescence microscopy. Cells pretreated with pcDNA3.1 (1 μ g) and pcDNA3.1-AEG-1 (1 μ g) were incubated with 10 μ M DYT-40 and vehicle, respectively, for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS at 1-hour intervals, permeabilized with 0.5% Triton X-100, and blocked with 2% BSA for 30 min, followed by incubation with primary antibodies (diluted 1:50) against AEG-1 and NF-κ B overnight at 4 °C. Subsequently the nuclei were stained with 4′ ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min prior to imaging. A FV10-ASW laser scanning confocal microscope [Ver 2.1] (Olympus Corp, MPE FV1000) was used for co-localization analysis 23  Orthotopic glioma model. Prior to implantation, 85-90% confluent C6 cells were trypsinized, rinsed with F12K+ 10% fetal calf serum, and centrifuged at 1000 rpm for 4 min. The cell pellet was resuspended in F12K and placed on ice. The concentration of viable cells was adjusted to 1 × 10 8 cells/mL in F12K. Each rat was anesthetized by a peritoneal injection with 0.4 mL/100 g of a 1% pentobarbital sodium solution and placed in a stereotactic frame (RWD Life Science, Shenzhen, China). After shaving and disinfection of the skin, the skull was exposed using a sagittal incision, and a burr hole 1 mm anterior and 3 mm lateral relative to the bregma was produced using a small drill. The cell suspension was injected into a depth of 6 mm from the skull surface, using a 2-μ L Hamilton (#2701) syringe (Reno, NV, USA) with a 26s-gauge needle mounted on a stereotactic holder 29 . After injection, the needle was left in place for 5 min and slowly withdrawn. The scalp incision was subsequently closed with surgical sutures. The animals were intramuscularly injected with 0.1 mL/rat of 80 U/mL benzylpenicillin sodium solution to prevent infection and returned to their home cages. The rats were divided into five groups. The negative control group was treated with vehicle. The positive control was intravenous (i.v.) injected with 10 mg/kg temozolomide. The other three groups were intravenously injected with DYT-40 (5, 10, 20 mg/kg) at 24 h after surgery, respectively. All drugs were administered once a day and continued for two weeks. The maximum cross-sectional area of the intracranial glioblastoma xenografts was determined by computer-assisted image analysis using a Leica Quantimet 500-system (Leica, Hamburg, Germany). The tumor volume was calculated as the square root of the maximal tumor cross-sectional area 3,30 . Histology, immunohistochemistry assay. The animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were removed from the skulls and post-fixed overnight at 4 °C in 4% paraformaldehyde. On the next day, the brains were transferred to 30% sucrose in PBS solution for 48 h at 4 °C. Coronal sections with a thickness of 10 mm were cut using a cryostat microtome (Leica CM1900, Germany). Hematoxylin and eosin (H&E) staining was used to visualize the tumor area and tumor necrosis. To evaluate cell proliferation, immunostaining for AEG-1 was used. The antibody against AEG-1 was diluted 1:300 in blocking solution containing 0.3% Triton X-100 and incubated overnight at 4 °C. After washing in PBS, the sections were incubated with a biotinylated secondary antibody for 2 h, followed by washing and further incubation with a streptavidin-biotin-peroxidase complex (Vector Laboratories). Apoptosis was examined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method (In Situ Cell Death Detection Kit; Roche Molecular Biochemicals) according to the manufacturer's instructions.
Statistical analysis. All statistical analyses were performed using SPSS 10.0 for Windows software package (SPSS Inc., Chicago, IL, USA). The data are represented as the mean ± SEM of three independent experiments. Statistical comparisons of the results were performed using analysis of variance. * ,# p < 0.05 compared with control; ** ,## p < 0.01 compared with control. $$,&& p < 0.01 compared with DYT-40 (10 μ M)-treated cells.   Fig. S2, temozolomide inhibited cell growth in C6 cells in a concentration-and time-dependent manner. The inhibitory effects were apparent at concentrations of 1.25, 2.5, 5, 10 and 20 μ M of temozolomide. The IC 50 values of temozolomide in C6 cells were 12.90, 11.16 and 6.82 μ M for 24, 48 and 72 h treatment, respectively. Thus, the efficacy of DYT-40 on glioma C6 cells growth seems to be better compared to temozolomide.
We evaluated the expression of the apoptotic protein Bax, the anti-apoptotic proteins Bcl-2 and Mcl-1, caspases 3, 9 and 8, and PARP using western blotting. The results showed that Bax expression increased while expression Bcl-2 decreased. Thus, the ratio of Bax/Bcl-2, which is crucial for the activation of the mitochondrial apoptotic pathway, increased in cells treated with DYT-40. In addition, Mcl-1, an anti-apoptotic Bcl-2 homolog, decreased after DYT-40 treatment (Fig. 3e). As shown in Fig. 3f, PARP cleavage increased as the concentration of DYT-40 increased. We further examined the involvement of caspases in DYT-40-mediated apoptosis. Compared with the controls, caspase-3, caspase-9 and caspase-8 were all activated after DYT-40 treatment for 24 h. These results demonstrated that the intrinsic apoptotic pathway is involved in DYT-40-induced apoptosis in U251 and U87 cells and that antitumor activity might be ascribed to apoptosis induction.

DYT-40 suppressed the invasion and epithelial-mesenchymal transition of malignant glioblastoma cells.
Tumor cell adhesion to the ECM and basement membrane was considered as a fundamental step for cancer metastasis. We examined the influence of DYT-40 on the adhesion of U251 and U87 cells to substrates that were precoated with fibronectin, which is an important component of the ECM. DYT-40 treatment (2.5, 5 and 10 μ M) suppressed U251 and U87 cell adhesion to fibronectin by 14.22 ± 9.57%, 41.76 ± 3.99 and 51.16 ± 6.87% and by 11.92 ± 4.32%, 25.93 ± 4.01 and 44.89 ± 3.41%, respectively (Fig. 4a). Moreover, the effects of DYT-40 (2.5, 5 and 10 μ M) treatment for 24 h on the invasion of cells were determined. pcDNA3.1-AEG-1 was transfected into U251 and U87 cells for 24 h, and the cells were treated with DYT-40 (10 μ M) for an additional 24 h. Subsequently, the cells were seeded onto the upside of transwell coated with matrigel, and the cells that invaded through the polycarbonate membrane were stained using crystal violet after 24 h of incubation. As shown in Fig. 4b, DYT-40 treatment reduced the invasion of U251 and U87 cells in a concentration-dependent manner. Cell invasion was rescued by pcDNA3.1-AEG-1 transfection.
Previous studies have shown that MMP-2 and MMP-9 play a critical role in cancer cell invasion by stimulating the degradation of the ECM 31 . The protein levels of MMP-2 and MMP-9 in DYT-40-treated U251 and U87 cells were decreased 25.75 and 58.54% (U251), 61.32 and 50.40% (U87), respectively, suggesting that DYT-40 suppresses the invasion ability of U251 and U87 cells by down-regulating the activity and expression of MMP-2/9 (Fig. 4c). To further investigate the effect of DYT-40 on malignant cell invasion, we examined the changes in epithelial and mesenchymal markers in DYT-40 treatments from 2.5 to 10 μ M at 24 h. As shown in Fig. 4d, DYT-40 increased the E-cadherin protein levels, whereas the N-cadherin and vimentin protein levels were decreased in U251 and U87 cells. Compared with the control groups, 10 μ M DYT-40 increased the E-cadherin levels by 159.69 and 174.68%. In contrast, the N-cadherin and vimentin protein levels were reduced 12.59 and 20.56% (U251) and 19.53 and 9.85% (U87), respectively.

DYT-40 suppressed AEG-1/NF-κB, PI3K/AKT and MAPK pathways. Nuclear factor kappa B
(NF-κ B) exerted an anti-apoptotic effect via the induction of genes that inhibit apoptotic signaling pathways 32 . In addition, AEG-1 induced the invasiveness of tumor cells and increased the expression of adhesion molecules through the activation of the NF-κ B signaling pathway 8 . AEG-1 also physically interacted with p65 to modulate the function of this protein in the nucleus 12 . The activation and translocation of p65, which is the functional active subunit of NF-κ B, were analyzed when U87 cells were treated with DYT-40. The nuclei were isolated, and the amount of NF-κ B p65 in the nuclear fraction was quantified by western blotting. As shown in Fig. 5a, the level of NF-κ B p65 protein in the nucleus was decreased after treatment with 10 μ M DYT-40 for 3, 6, 12, 18 and 24 h. Moreover, the total amount of NF-κ B p65 decreased after DYT-40 treatment (Fig. 5b). Because NF-κ B activation resulted from the rapid phosphorylation, ubiquitination and, ultimately, proteolytic degradation of Iκ B 33 and because IKK is required for the phosphorylation of Iκ B 34 , the phosphorylation levels of IKKα /β and Iκ Bα were detected. DYT-40 (12, 18 and 24 h) efficiently inhibited the phosphorylation of IKKα /β and Iκ Bα , whereas the total steady-state levels remained unchanged (Fig. 5b).
For certification, pcDNA3.1 and pcDNA3.1-AEG-1 were transfected into U87 cells for 24 h, and the cells were treated with DYT-40 (10 μ M) for an additional 24 h. The nuclei were isolated, and the amount of NF-κ B p65 in the nuclear fraction was quantified using western blotting. As shown in Fig. 5c, the level of NF-κ B p65 protein in the nucleus was evidently decreased after treatment with 10 μ M DYT-40 for 24 h, and this effect was rescued by AEG-1 over-expression. The inhibition of both the distribution of AEG-1 and nuclear translocation of NF-κ B p65 after DYT-40 (10 μ M) treatment were observed using immunofluorescence confocal microscopy. This inhibition was rescued by AEG-1 over-expression (Fig. 5d).
As shown in Fig. 5e, DYT-40 treatment decreased the phosphorylation of AKT, and ERK kinase with no change in the total steady state protein levels. Furthermore, PI3K (p110) and PI3K (p85) was down-regulated and the tumor gene C-myc was down-regulated after treatment with DYT-40, suggesting that DYT-40 inhibited AEG-1 expression, which is involved in the suppression of the phosphorylation of PI3K/AKT and the MAPK axis. For certification, pcDNA3.1 and pcDNA3.1-AEG-1 were transfected into U87 cells for 24 h, and the cells were treated with DYT-40 (10 μ M) for an additional 24 h. The inhibition of both the phosphorylation of AKT, and ERK kinase after DYT-40 (10 μ M) treatment was rescued by AEG-1 over-expression (Fig. 5f). As shown in Fig. S4, DYT-40 inhibited the expression of AEG-1, C-myc, NF-κ B p65, Iκ Bα and p-Iκ Bα in C6 cells in a concentration-dependent manner.   times (0, 12, 18, and 24 h). The phosphorylation levels of IKKα /β and Iκ Bα was detected by Western blotting using specific antibodies where GAPDH was used as loading control. (c) The pcDNA3.1 (1μ g) and pcDNA3.1-AEG-1 (1 μ g) were transfected into U87 cells for 24 h, and cells were treated with DYT-40 (10 μ M) for an additional 24 h. After the isolation of nucleus and cytoplasm extracts, the levels of protein were measured by Western blotting. *p < 0.05 and ** p < 0.01. (d) Nuclear translocation of NF-κ B p65 and expression of AEG-1 were determined by triple-label immunofluorescence confocal microscopy. The cells were fixed and stained with rabbit anti-AEG-1, followed by Alexa Fluor 568-conjugated anti-rabbit antibody (red); NF-κ B p65 protein expression was stained with rabbit anti-NF-κ B p65 antibody (green) and secondary antibody goat anti-rabbit IgG/FITC. The nuclei were visualized by staining with DAPI (blue). The merged images represent the areas within the yellow squares and highlight the relative location of NF-κ B p65 to AEG-1.  (Fig. 6a). We did not find obvious pathological changes in rats after DYT-40 treatment. The DYT-40-treated animals showed a dramatic reduction in the tumor volume at 14 days of treatment compared with vehicle-treated animals (Fig. 6b). Vehicle-treated animals exhibited a median tumor volume of 530.08 mm 3 ± 43.99, whereas 5, 10 and 20 mg/kg DYT-40-treated animals revealed tumor volumes of 447.38 mm 3 ± 62.75, 254.61 mm 3 ± 55.55, 190.08 mm 3 ± 64.01, reflecting 16%, 52% and 64% reduction, respectively. Temozolomide treatment reduced tumor volumes 36% after 14 days (Fig. 6b). Moreover, the results of H&E staining revealed that the glioma model rat treated with DYT-40 exhibited better defined tumor margins and fewer invasive cells to the contralateral striatum compared with vehicle control and temozolomide-treated rats (Fig. 6c). As shown in Fig. 6d, the microscopic examination of AEG-1-stained tumor sections shows a decrease in AEG-1-positive cells in DYT-40-treated rats compared with the untreated controls. The in vivo apoptotic response of glioma cells to DYT-40-treatment was investigated by TUNEL staining. The microscopic examination of the tumor sections showed that compared with the untreated controls, DYT-40-treatment increased the number of TUNEL-positive cells.

Discussion
Since the discovery of AEG-1, many studies have focused on AEG-1 to expand the knowledge of this important molecule 35 . The crucial role of AEG-1 ranges from cancer biology to the molecular mechanisms underlying the biological functions. AEG-1 can be defined as a multifunctional oncogene that is over-expressed in complex types and progressive human cancers 6,10,11,36 . The elevated expression of AEG-1 in tumor cells enhanced the phenotypic characteristics of malignant aggressiveness, including increased robust proliferation, migration and invasion to surrounding tissues, neovascularization, and enhanced chemoresistance. The diverse functions of AEG-1 during tumor progression in various cancers including brain tumorigenesis have been elucidated. However, studies of AEG-1 inhibitors are urgently needed for clinical therapy. Recent studies have provided definitive evidence that elevated AEG-1 expression is a common event in brain tumors of diverse origin, including GBM 4 . In the present study, we focused on the regulatory mechanism of AEG-1/MTDH-mediated autophagy during malignant glioma EMT and invasion. An associated study has been published online in Oncotarget 37 . The results provided evidence that AEG-1 enhances human malignant glioma susceptibility to TGF-β 1-triggered EMT via autophagy induction, which is associated with glioblastoma development and progression.
Several groups have demonstrated the diverse function of AEG-1 during the development and progression of cancers. The generalization of the signaling cascades associated with AEG-1 includes (1) the activation of the transcription factor nuclear factor κ B (NF-κ B), partially through direct interaction with p65, a TNF-α downstream signaling component, is associated with several human diseases, including cancer, and NF-κ B controls the expression of multiple genes involved in tumor progression, invasion, and metastasis 8 ; (2) enhancement of phosphorylation of MAPK molecules, including ERK1/2 and p38, which subsequently activates Wnt/β -catenin signaling and consequently results in tumor angiogenesis 10 ; (3) suppressing apoptosis through a systematic mechanism that up-regulates apoptosis inhibitors via the indirect activation of the PI3K/AKT signaling pathway 19 ; (4) recruitment of oncogene C-myc to the AEG-1 promoter region resulting from the activation of the Ras oncogene, which consequently activates AEG-1 expression 7 ; (5) interaction with staphylococcal nuclease domain-containing 1 (SND1) and enhancing the activity of RNA-induced silencing complex (RISC), which leads to degradation of target of onco-miRNAs tumor suppressor mRNAs 38 ; and (6) triggering protective autophagy, which is a common mechanism employed by tumor cells to adapt with metabolic stress 39 . In addition, we examined the mechanism that AEG-1/MTDH enhances human malignant glioma susceptibility to TGF-β 1-triggered epithelial-mesenchymal transition via autophagy induction.
The inhibition of cancer cell proliferation by anticancer chemotherapeutic drugs is normally attributed to apoptosis, which is characterized by cytoplasmic shrinkage, chromatin condensation, and DNA fragmentation 40 . Apoptosis is an active form of programmed cell death that occurs in response to specific treatment, including mitochondria-mediated endogenous apoptosis and death receptor-mediated exogenous apoptosis [41][42][43] . As a consequence of adduct formation, DNA repair was simultaneously activated with cell cycle arrest. The failure to repair DNA led to cell apoptosis 44 . Mitochondria-mediated endogenous apoptosis is characterized by the decreased expression of apoptosis inhibitor proteins of the Bcl-2 gene family, including Bcl-2 and Mcl-1 and increases in the expression of the apoptotic protein Bax 45,46 . The involvement of caspase-8 and caspase-9 in apoptosis has been demonstrated by the cleavage of caspase-3 and PARP and the fragmentation of the caspase-3 substrate 46,47 . Thus, the members of both caspases and the Bcl-2 family as well as other proteins and events, such as oxidative stress and cell cycle arrest, could be defined as determinants of apoptosis 48 .
EMT is a complex biologic reprogramming that is considered a crucial event in metastasis and tumor progression 49,50 . At the early metastatic stage of tumors, the main feature of EMT occurrence is characterized by the loss of epithelial phenotype (E-cadherin) and acquisition of motility, invasive potential and mesenchymal characteristics (N-cadherin and vimentin) 51 . In addition, β -catenin, which originally combines with E-cadherin at the intracellular C-terminus, exhibits nuclear translocation and release from the membrane to promote a series of invasive molecules 52 . Simultaneously, AEG-1 activates Wnt/β -catenin signaling, which triggers EMT in some malignant tumors. As a result of the crucial role for the EMT in metastasis, suppressing the EMT checkpoint and progression in cancers has been recently considered a promising strategy to inhibit metastasis and prolong the survival of patients undergoing cancer.
The results of the present study have designed DYT-40, a novel synthetic (E)-2-(2-(4-chlorostyryl)-5-nitro-1H-imidazol-1-yl)ethyl-2-(2,3-dihydrobenzo[b] [1,4]dioxin-6-yl) acetate, which significantly blocks malignant glioblastoma growth and invasion via the inhibition of AEG-1 expression. Moreover, the inhibition of AEG-1 expression significantly induces the apoptosis and suppresses the invasive ability of human malignant glioblastoma U251 and U87 cells by modulating MMP-2, MMP-9 activity and EMT characteristics. The activation and translocation of p65, the functional active subunit of NF-κ B, were analyzed in U87 cells treated with DYT-40. The nuclei were isolated, and the amount of NF-κ B p65 in the nuclear fraction was quantified by western blotting. The level of NF-κ B p65 protein in the nucleus was evidently decreased after treatment with 10 μ M DYT-40 for 3, 6, 12, 18, and 24 h. DYT-40 treatment decreased the phosphorylation of AKT and ERK kinase, with no change in the total steady state protein level. Furthermore, PI3K (p110) and PI3K (p85) and the tumor gene C-myc were down-regulated when treated with DYT-40, suggesting that DYT-40 inhibited AEG-1 expression, which is involved in the suppression of the phosphorylation of PI3K/AKT and MAPK.
In summary, these results indicated that DYT-40 inhibited the proliferation and invasion of malignant glioblastoma cells in a concentration-dependent manner. AEG-1 inhibition by DYT-40 was involved in the MAPK and PI3K/AKT-NF-κ B signal transduction pathway and led to DYT-40-induced apoptosis and DYT-40-inhibited EMT in U251 and U87 cells. The inhibition of AEG-1 expression induced by DYT-40 resulted in the down-regulation of NF-κ B expression, which exhibits a key molecular mechanism in which DYT-40 inhibits cell growth and invasion in malignant glioblastoma cells. To enter the brain, drugs must pass through the endothelial cells of the capillaries of the central nervous system (CNS) or be actively transported. Lipid-soluble drugs readily penetrate into the CNS because they can dissolve in the membrane of the endothelial cells (Page 554 of Lippincott's Illustrated Reviews Pharmacology). DYT-40 is a synthetic derivative of nitroimidazole that can cross blood brain barrier because of its lipid-solubility. Accordingly, DYT-40 could penetrate the blood brain barrier following intravenous administration. In addition, DYT-40 with a low molecular weight of 455.85 has an enhanced ability to cross the blood-brain barrier even in the presence of meningeal inflammation. Actually, transport experiment of 125 I-labeled DYT-40 on rat brain microvascular endothelial cells (rBMEC) would be more meaningful 53 .
Therefore, the inhibition of AEG-1 by the designed compound DYT-40 molecular targeting to AEG-1 might provide a novel strategy for the treatment of malignant glioblastoma cells. The results of the present study produced several important conclusions to further investigate the more potential role of AEG-1 in physiological or pathological processes and the sensitivity of cancer cells in response to clinically chemotherapeutic agents.