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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 4  |  Page : 889-898

Anticancer effects of FL34 through the inhibition of GLI1 in glioblastoma


1 Department of Pharmacology, Institute of Material Medical, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
2 Department of Synthetic Medicinal Chemistry, Beijing Key Laboratory of Active Substance Discovery and Drug ability Evaluation, Institute of Material Medical, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
3 Department of Neurosurgery, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

Date of Web Publication14-Aug-2019

Correspondence Address:
Xueji Li
Department of Neurosurgery, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021
China
Yan Li
Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_564_18

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 > Abstract 


Background: The hedgehog (HH) signaling pathway is abnormally activated in glioblastoma (GBM); thus, its downstream effector GLI1 may be a suitable target for the treatment of GBM. The aim of the present study was to evaluate the antitumor activities of a novel compound, FL34, in GBM through the inhibition of GLI1.
Methods: The effect of FL34 on suppressing the proliferation, angiogenesis, and invasion of GBM cells was investigated in vitro using proliferation, invasion, tube formation, flow cytometry, GLI1 dual luciferase, reverse transcription-quantitative polymerase chain reaction, and western blot assays. A subcutaneously transplanted and orthotopic U-87 MG GBM cell xenograft model was used to study the effect of FL34 on tumor growth in vivo.
Results: The results of the present study demonstrated that FL34 markedly inhibited the proliferation, invasion, and angiogenesis of GBM, in addition to decreasing the transcriptional activity and expression of GLI1, resulting in the downregulation of GLI1 target genes, including B-cell lymphoma-2, vascular endothelial growth factor, and matrix metalloproteinases. Furthermore, FL34 inhibited the activation of GLI1 without influencing upstream canonical HH/Smoothened signaling or through crosstalk with other oncogenic pathways, including Ras/ERK and AKT signaling. At a dose of 30.0 mg/kg, FL34 suppressed tumor growth by 78.74% in tumor weight in subcutaneously transplanted U-87 MG xenograft models and by 64.24% in volume in orthotopic U-87 MG GBM xenograft models.
Conclusions: These data suggested that FL34 exerted antitumor activity mediated by the inhibition of GLI1 and that FL34 may be a potential antitumor candidate compound that could be used to develop new antitumor drugs for the treatment of GBM.

Keywords: Antitumor, glioblastoma, GLI1, hedgehog


How to cite this article:
Li Y, Tang K, Huang L, Liu C, Du Q, Li T, Yan C, Feng Z, Li X. Anticancer effects of FL34 through the inhibition of GLI1 in glioblastoma. J Can Res Ther 2019;15:889-98

How to cite this URL:
Li Y, Tang K, Huang L, Liu C, Du Q, Li T, Yan C, Feng Z, Li X. Anticancer effects of FL34 through the inhibition of GLI1 in glioblastoma. J Can Res Ther [serial online] 2019 [cited 2019 Sep 18];15:889-98. Available from: http://www.cancerjournal.net/text.asp?2019/15/4/889/264292




 > Introduction Top


Glioblastoma (GBM) is the most common and malignant type of central nervous system tumor in humans.[1],[2] At present, surgical resection followed by radiation and chemotherapy is the standard treatment for GBM.[3] Patients with GBM have a poor prognosis and a low long-term survival rate, despite efforts to identify the molecular basis of GBM tumorigenesis and develop novel cancer therapies. Furthermore, chemoresistance and recurrence are frequent.[3] Therefore, effective targeted therapeutic strategies are urgently required.

The hedgehog (HH) signaling pathway may be initiated by the three HH ligands: sonic HH (SHH), Indian HH or Desert HH. Among them, SHH is the most widely expressed. The binding of HH ligands to patched (PTCH), a 12-pass transmembrane protein that inhibits smoothened (SMO),[4] relieves SMO from the suppression of PTCH. The stimulation of SMO results in the activation of downstream signaling targets, including transcription factors in the glioma-associated oncogene homolog family, for example, GLI1-3 and GLI target genes.[5]

The HH pathway plays an important role in the control of cell proliferation, differentiation, angiogenesis, and tumor cell invasiveness.[6],[7] Based on the aberrant activation of the HH signaling pathway in multiple malignant tumors, including GBM, skin, lung and pancreatic cancer, the inhibition of the HH pathway by new therapeutic approaches has been investigated in various solid tumors.[8],[9]

At present, HH modulators in clinical therapy have focused on the cell membrane receptor SMO, including GDC-0449 (vismodegib) and NVP-LDE225 (erismodegib). These drugs are selective, orally bioavailable SMO inhibitors that inhibit tumor growth and survival through antagonism of the SMO receptor.[10] However, resistance to SMO antagonists frequently emerges due to germline or acquired SMO mutations,[11],[12],[13],[14] or through SMO-independent means, including the activation of downstream GLI [14],[15],[16] or the upregulation of noncanonical GLI signaling. Furthermore, GLI1 has been reported to be hyperactivated in GBM.[16],[17] Thus, GLI1 may represent an attractive target for the development of antitumor drugs to treat GBM.

The present study showed the inhibitory effects of FL34 on GBM cell growth, invasion, and angiogenesis in vitro and subcutaneously transplanted, orthotopic xenograft models. Furthermore, FL34 decreased the transcriptional activity and expression of GLI1, resulting in the downregulation of GLI1 target genes, including B-cell lymphoma-2 (Bcl-2), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), without influencing the upstream SMO and cross-linked Ras/ERK and AKT signaling pathways. The results of the present study suggested that FL34 may be a potential antitumor candidate compound for GBM tumors.


 > Methods Top


Cell lines and culture

U-87 MG human brain tumor and HUVEC-T (HUVEC-T cells are SV40T-transformed HUVEC cells) cell lines were obtained from the Cell Center of the Chinese Academy of Medical Sciences (CAMS) and Peking Union Medical College (PUMC. The T98G GBM cell line was obtained from Shanghai Xiangf Bio Company. U-87 MG cells were cultured in minimal essential media (MEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 IU/mL penicillin, and 100 μg/mL streptomycin. Other cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.,) supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.

Drugs and compounds

FL34, a small molecule compound named N-(2-(2-(2-ethoxyethoxy) ethoxy)-5-(1H-benzo[d]imidazol-2-yl)phenyl) but-2-ynamide (CN201410208409.2) and temozolomide (TMZ) was synthesized by the Department of Pharmacochemistry of the Institute of Materia Medica of the CAMS and PUMC (high-performance liquid chromatography [HPLC] purity >98%). For the in vitro experiments, FL34 was dissolved in dimethyl sulfoxide (DMSO) and stored at 4°C until use. DMSO was used as the vehicle control in all in vitro experiments at a final concentration of 0.1%. For in vivo experiments, FL34 and TMZ were dissolved in a solution of 20% PEG400.

Cell proliferation assay

Cell proliferation was analyzed by MTT assay. Cells were plated onto 96-well plates and cultured for 24 h. The cells were treated with various concentrations of FL34 in triplicate. At 72 h, 20 μl MTT (5.0 mg/mL) was added and the cells incubated for a further 4 h at 37°C in a 5% CO2 atmosphere. The solution was removed and 150 μl DMSO was added. The optical density at 570 nm was measured using an ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiments were performed in triplicate.

Apoptosis assay

Apoptosis was analyzed using an Annexin V-FITC Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., China). U-87 MG and T98G cells were plated onto 6-well plates and cultured for 24 h before FL34 was added at different concentrations. Following incubation for 72 h, the cells were harvested and washed with cold phosphate-buffered saline (PBS). Subsequent to suspension in binding buffer, the cells were stained with 10 μL FITC annexin V and 5 μL propidium iodide (PI) for 15 min. The cell samples were analyzed using an ACCURI C6 flow cytometry system (BD Biosciences, US).

Cell invasion assay

An invasion assay was performed using Transwell inserts containing polycarbonate filters with 8.0-μm pores. Matrigel (10 μL, 1.0 mg/mL) was added to the upper chamber. The cells (2 × 105 cells/well) were suspended in 200 μL 0.1% bovine serum albumin with or without different concentrations of FL34 and seeded into the upper chamber; 600 μL normal medium was placed in the lower chambers. Following 14 h incubation at 37°C in a 5% CO2 atmosphere, the cells on the upper surface of the membrane were mechanically removed and the invading cells on the lower surface were fixed with methanol and stained with hematoxylin and eosin. The numbers of invasive cells in five random fields per membrane were counted at ×200 magnification (Olympus IX70, Japan). The experiment was performed in triplicate.

Tube formation assay

A total of 5000 HUVEC-T cells, treated or untreated with FL34, were seeded into a 96-well plate pre-coated with Matrigel. Following incubation at 37°C for 5 h, the cells were examined for capillary-like tube formation and photographed under a microscope (×100 magnification, Olympus IX70). All experiments were performed in triplicate.

In vivo efficacy evaluation using a xenograft model

Procedures for the animal study were performed with the approval of the Animal Care and Use Committee of the CAMS and PUMC. Female 6-week-old BALB/c/nu nude mice (Vital River Laboratories) were used for the xenograft study. U-87 MG cells (1 × 107) were subcutaneously injected into the left flank of each mouse. When tumor volumes reached an average of 200 mm 3, the tumor-bearing mice were randomly separated into a control group and three treatment groups, each containing five animals. The mice in the control group received an oral formulation of vehicle consisting of 20% PEG400. Treatments were administered orally and included 15.0 or 30.0 mg/kg FL34 or 30.0 mg/kg TMZ daily (FL34 14 times; TMZ 5 times). The mice were weighed, the tumor sizes were measured using Vernier calipers, and the tumor volume was calculated (tumor volume [mm 3] = [width × width × length]/2) twice each week. The relative tumor volumes (RTVs) were calculated as the measured tumor volume each day (Vt) divided by the starting volume (V0). The mice were sacrificed at the end of the treatment period. The tumors were measured and weighed, and images were captured. Each tumor was stored at −80°C until further analyses.

Orthotopic U-87 MG glioblastoma xenograft model and magnetic resonance imaging assessment of tumor volumes

The heads of anesthetized male BALB/c/nu nude mice (Beijing Huafukang bioscience Co., Inc., China) were immobilized, and a 1-mm hole was drilled in the skull 1 mm posterior and 2 mm lateral to the bregma, on the right side. U-87 MG cells (1 × 106 cells in 5 μL PBS) were implanted intracerebrally 2.5 mm below the skull surface using a 26-gauge needle. Following injection, the skin was closed with surgical sutures. The animals were divided into three groups (control, FL34, and TMZ) with six mice per group. Treatments were started on the 5th day postsurgery. FL34 and TMZ were administered orally at a dose of 30.0 mg/kg every day. FL34 was administered 6 days/week, and TMZ was successively administered for 5 days. The control mice received the vehicle alone.

MRI experiments were performed on a 7 Tesla 16 cm horizontal-bore magnet imaging system (PharmScan 70/16 US, Bruker, Switzerland). The animals were anesthetized with isoflurane (3% induction, 2% maintenance at 1 l/min in oxygen). A mouse surface head coil was used for signaling detection, and a 72-mm quadrature volume coil was used for transmission. Multislice T2-weighted image (T2WI) images were collected using a T2_Turbo RARE sequence with the following parameters: TR/TE, 2500 ms/35 ms; rare factor, 8; image size, 256 × 256; field of view, 25 mm × 25 mm; and slice thickness, 0.5 mm. Morphological T2WI images were used to assess tumor growth and calculate the tumor volumes. The tumor volumes were calculated from multiple MRI slice datasets using Philips DICOM Viewer 3.0 software (Royal Dutch Philips Electronics Ltd., Amsterdam, The Netherlands).

GLI1 dual-luciferase assays

The luciferase reporter plasmid containing the GLI1-binding site (pGMGLI1-Lu) and the TK-Renilla luciferase plasmid (pGMR-TK) were purchased from Genomeditech Shanghai Co., Ltd. U-87 MG cells in 96-well plates were cotransfected with pGMR-TK and pGMGLI1-Lu using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.,) according to the manufacturer's protocol. At 24 h after transfection, the cells were treated with the indicated doses of FL34 and incubated for a further 48 h. Luciferase assays were performed using a dual luciferase reporter assay system (Promega Corporation; cat. no. E1960) following the manufacturer's protocol. The firefly luciferase values were normalized to the Renilla values.

Reverse transcription-quantitative polymerase chain reaction

The Total RNA was isolated from U-87 MG and T98G cells treated with different concentrations of FL34 for 48 h and from U-87 MG xenograft tissues (3–4 randomly-selected tumors from U-87 MG xenograft mice from the control, 15.0, and 30.0-mg/kg FL34 groups) using TRIzol reagent (Takara, Dalian, China), following the manufacturer's protocol. The total RNA was reverse transcribed to cDNA using the SuperQuick RT MasterMix kit (CWBIO, Beijing, China). The quantitative polymerase chain reaction (qPCR) analyses were performed using UltraSYBR Mixture (CWBIO) on a 7900 Real-Time PCR system (Applied Biosystems) using the following primers: forward, 5'-CAGTGTGGGGACAGAAGGA-3' and reverse, 5'-CGGGGAGAAGAAAAGAGTGG-3' for GLI1; forward, 5'-CACATGGCCTCCAAGG-3' and reverse, and 5'-GGTTGAGCACAGGGTA-3' for GAPDH. The mRNA level of GLI1 was normalized to GAPDH, and the fold change of relative mRNA expression levels was determined using the 2− ΔΔ Ct method.

Western blot analysis

Protein extraction and western blotting were performed as previously described.[18] Pooled tissues from 2 to 3 randomly-selected tumors from U-87 MG xenograft mice from the control, 15.0 mg/kg and 30.0 mg/kg FL34 groups, and U-87 MG and T98G cells treated with various concentrations of FL34 for 72 h, were lysed. For western blotting, the protein concentration was determined using the bicinchoninic acid method. Total proteins (50 μg) were separated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane by semi-wet electrophoresis. The membranes were blocked with Tris-buffered saline containing 1% Tween 20 and 5% skimmed dry milk, and incubated with dedicated primary antibodies (dilution, 1:1000; Cell Signaling Technology, Inc., Shanghai, China; Abcam Trading (Shanghai) Company Ltd., Shanghai, China.) overnight at 4°C. The samples were detected with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (dilution, 1:5000; Santa Cruz Biotechnology, Inc.,) for 1 h at room temperature, and developed using an enhanced chemiluminescence Western blot detection and analysis system (Applygen Technologies, Inc.). β-actin was used as a loading control.

Statistical analysis

Data were expressed as means ± standard deviation (SD). Differences between means were assessed using Student's two-tailed t-tests. P ≤ 0.05 was considered statistically significant. Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA) was used for the statistical analyses.


 > Results Top


FL34 inhibition of glioblastoma cell proliferation

The growth inhibitory effect of FL34 was evaluated in U-87 MG and T98G cell lines using an MTT assay. A concentration-dependent inhibition in cell viability was observed following treatment with FL34 for 72 h, with IC50 values of 44.3 ± 2.88 and 79.6 ± 8.75 nmol/L for U-87 MG and T98G cells, respectively. Doses of 0.5, 5.0, and 50.0 nmol/L FL34 were selected for U-87 MG cells and 1.0, 10.0 and 100.0 nmol/L for T98G cells.

FL34-induced apoptosis in U-87 MG and T98G cells

FL34 was examined for its effect on the rate of apoptosis by annexin-V-PI double staining. The early and late apoptotic rates were 7.63% and 1.94%, respectively, in U-87 MG cells; and the early and late apoptotic population tended to increase after 72 h of 5.0-50.0 nmol/L FL34 administration (early apoptotic population: 30.96 and 60.74%; late apoptotic population: 2.58 and 4.27%, for 5.0 and 50.0 nmol/L FL34, respectively); [[Figure 1]ai and ii]. Similar results were observed in T98G cells, although a higher FL34 concentration (10.0–100.0 nmol/L) was required. Following treatment with various concentrations of FL34 for 72 h, the populations of early and late apoptotic cells increased in a concentration-dependent manner, particularly, those of the early apoptotic cells (26.66 and 35.00% in the 10.0 and 100.0 nmol/L FL34-treated groups, compared to 5.38% in the control group); [[Figure 1]bi and ii]. These results suggested that FL34 induced apoptosis in a dose-dependent manner in GBM cells.
Figure 1: The effect of FL34 on the apoptosis of glioblastoma cells. (a) FL34 treatment-induced apoptosis of U-87 MG cells. Cells were incubated with FL34 for 72 h, stained with annexin-V-FITC/PI, and analyzed by flow cytometry. The flow cytometry results are shown as quantitative bar graphs in which the error bars indicate standard deviation. *P < 0.05, **P < 0.01 versus control cells (ai), Representative scatter diagram of apoptosis after annexin-V-FITC/PI double staining analyzed by flow cytometry (aii); (b) FL34 treatment-induced apoptosis of T98G cells. Cells were incubated with FL34 for 72 h, stained with annexin V-FITC/PI, and analyzed by flow cytometry. The flow cytometry results are shown as quantitative bar graphs, in which the error bars indicate standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 versus control cells (bi); Representative scatter diagram of apoptosis after annexin-V-FITC/PI double staining analyzed by flow cytometry (bii)

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FL34 suppression of U-87 MG and T98G cell invasion

To evaluate the ability of GBM cells treated with various concentrations of FL34 to invade a reconstituted basement membrane, a Transwell invasion assay was performed. U-87 MG and T98G cells were treated with DMSO or FL34 at doses of 0.5–50.0 nmol/L in U-87 MG cells, and 1.0–100.0 nmol/L in T98G cells and were incubated for 14 h, a time point that did not affect GBM cell proliferation according to the MTT assay (IC50 values were 0.41 ± 0.077 μmol/L for U-87 MG and 3.51 ± 0.68 μmol/L for T98G cells following a 14-h incubation with FL34). FL34 had a dose-dependent effect on cellular invasion, with invasion inhibition rates of 43.3% and 55.7% in 5.0 and 50.0 nmol/L FL34-treated U-87 MG cells, respectively [[Figure 2]ai and ii]. Similarly, 10.0 and 100.0 nmol/L FL34 led to a marked inhibition of invasion, at 20.3 and 46.0%, in T98G cells [[Figure 2]bi and ii]. These data indicated that FL34 inhibited the invasion of U-87 MG and T98G cells in vitro.
Figure 2: Effect of FL34 on the invasion of glioblastoma cells and the tube formation of HUVEC-T cells in vitro. (a and b) Equal numbers of U-87 MG (ai and ii) and T98G (bi and ii) cells were seeded into the upper compartment of a Transwell system and allowed to invade through the Matrigel for 14 h. The invaded cells were fixed and counted. Decreased invasion is observed cells treated with FL34 compared to that in control cells. The error bars represent the standard deviation. *P < 0.05 and **P < 0.01 versus control cells. (c) Tube formation assay. The tube formation of HUVEC-T cells was inhibited following the administration of FL34 for 5 h. (ci) Representative pictures are presented; (cii) The results of total tubule length are shown as quantitative bar graphs, the error bars represent the standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, versus control cells

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FL34 inhibition of tube formation in HUVEC-T cells

The presence of blood vessels is associated with the malignancy and aggressiveness of glioma.[19] Thus, the present study examined the effects of FL34 on angiogenesis using a HUVEC-T cell tube formation assay. As presented in [Figure 2]ci and ii, the tube length was markedly decreased following treatment with 10.0 and 100.0 nmol/L FL34 for 5 h, whereas the proliferation of HUVEC-T cells was not influenced at the above concentrations (IC50: 11.65 ± 3.54 μmol/L at 5-h incubation).

FL34 inhibition of U-87 MG tumor growth

Following the results obtained from the in vitro study, the antitumor efficacy of FL34 on the growth of subcutaneously transplanted U-87 MG cells was investigated. When the volume of the tumors reached ~200 mm 3, nude mice were orally treated with 15.0 or 30.0 mg/kg FL34 or 30.0 mg/kg TMZ (n = 5 mice/group). The administration of FL34 significantly inhibited the growth of the tumor mass. At the end of the experiment, treatment with FL34 at 15.0 and 30.0 mg/kg resulted in tumor weight losses of 66.92% and 78.74%, respectively. In addition, FL34 treatment caused tumor volume decreases of 71.56% and 81.05%, respectively [Table 1] and [Figure 3]a, [Figure 3]b. All animals gained weight during the experiment, indicating that the concentrations of FL34 used were well tolerated [Table 1].
Table 1: Antitumor activity of FL34 in a subcutaneously transplanted U-87 MG xenograft model

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Figure 3: Inhibition of U-87 MG tumor growth by FL34 in the subcutaneously transplanted and orthotopic xenograft model. (a and b) The activity of FL34 was determined in the subcutaneously transplanted U-87 MG xenografts in nude mice. U-87 MG-bearing mice were randomly separated into four groups of five animals. One group received 20% PEG400 as a vehicle control; the other groups received 30.0 mg/kg temozolomide, or 15.0 or 30.0 mg/kg FL34 daily (FL34 14 times; temozolomide five times). The xenograft tumor volumes were measured. (a) Mean relative tumor volumes ± standard deviation at the indicated time points. (b) Corresponding U-87 MG tumors in the subcutaneously transplanted xenograft model. (c) Tumor volumes for U-87 MG cells in the orthotopic xenograft model. U-87 MG tumor-bearing animals receiving different treatments were assessed by MRI. There was a significant decrease in the tumor volume in mice treated with FL34 or temozolomide when compared with untreated mice. (d) T2 weighted images of U-87 MG glioma-bearing mice in situ. Each image was obtained from different mice from each treatment group. (di) representative control mouse (15 days following U-87 MG cells implantation); (dii) representative FL34-treated mouse (15 days following U-87 MG cells implantation)

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To further prove the effect of FL34 in vivo, an intracranial glioma model in nude mice was generated. Mice were intracranially injected with U-87 MG cells. The mice were treated with FL34 and TMZ at 30.0 mg/kg. MRI was performed to evaluate the activity of FL34 on orthotopic U-87 MG tumor growth. The FL34-treated group exhibited marked shrinkage in tumor volume, with an inhibition rate of 64.24% compared to the control group [Table 2] and [[Figure 3]c, [Figure 3]di and ii].
Table 2: Antitumor activity of FL34 on an orthotopic U-87 MG xenograft model

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FL34 influence on hedgehog signaling pathway activity

To investigate the effect of FL34 on GLI1 transcriptional activation, a GLI1 luciferase reporter assay was performed. U-87 MG cells were transfected with GLI1 firefly luciferase and Renilla luciferase plasmids, with or without various concentrations of FL34. The results demonstrated that FL34 dose-dependently inhibited GLI1-luciferase reporter activity, with an IC50 of 19.86 nmol/L [Figure 4]a, with an inhibition rate of 76.0% compared to that of the control after 48 h of treatment with 100.0 nmol/L FL34.
Figure 4: Influence of FL34 on HH signaling pathway activity. (a) GLI1 inhibitory activity of FL34 measured by a dual luciferase reporter assay. The GLI1 activity was inhibited following the administration of FL34 for 48 h. The error bars represent the standard deviation. *P < 0.05 versus control cells. (b) The effect of FL34 on the level of GLI1 in U-87 MG and T98G cells analyzed by reverse transcription-quantitative polymerase chain reaction. Data were normalized to GAPDH expression. Quantitative data are expressed as the mean ± standard deviation. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. (c) The effect of FL34 on the HH signaling pathway in U-87 MG and T98G cells analyzed by western blotting. Cells were treated with the indicated concentrations of FL34 for 72 h. “Con” refers to untreated control samples. Whole-cell lysates were subjected to western blotting with antibodies against the indicated proteins; western blotting for β-actin was used as a loading control. Representative western blot images are presented for each experiment. (d) The effect of FL34 on the HH signaling pathway in U-87 MG xenograft tumors. Mice bearing U-87 MG tumors were randomized and treated with FL34 15.0 or 30.0 mg/kg FL34 daily for 14 days. Left panel, GLI1 mRNA levels in U-87 MG xenograft tumors. The error bars represent the standard deviation, **P < 0.01. Right panel lysates of two or three mice from each group were pooled. Each lane represents one protein pool, and two or three pools/group was subjected to western blot analysis. β-actin was included as a loading control. The blots were incubated with the indicated antibodies. Representative blots are presented

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To further confirm the effect of FL34 on the HH pathway, U-87 MG and T98G cells treated with various concentrations of FL34 for 48 h were examined for GLI1 expression by reverse transcription-qPCR (RT-qPCR). Concomitantly, quantitative measurement demonstrated significantly downregulated GLI1 mRNA levels. FL34 doses of 5.0 and 10.0 nmol/L suppressed the expression of GLI1 by ~50% in both U-87 MG and T98G cells [Figure 4]b. Moreover, the inhibitory effect of FL34 on the HH pathway was further validated in U-87 MG and T98G cells by western blotting. The alterations in GLI1 protein expression levels exhibited similar trends to the alterations in mRNA levels. The results demonstrated a significantly decreased expression of GLI1 in U-87 MG cells treated with 5.0 and 50.0 nmol/L FL34 and in T98G cells treated with 10.0 and 100.0 nmol/L FL34 [Figure 4]c.

Simultaneous repression of GLI1 activity was confirmed by the reduction in the mRNA and protein expression levels of GLI1 by RT-qPCR and western blotting in the FL34-treated tumor groups [Figure 4]d. The mRNA level of GLI1 in tumors treated with 15.0 and 30.0 mg/kg of FL34 was decreased compared to that in tumors of the control in the xenograft model [Figure 4]d. Similar to the results of RT-qPCR, the protein expression of GLI1 was downregulated in tumors administered 15.0 and 30.0 mg/kg FL34, as analyzed by western blotting [Figure 4]d.

SMO is a GLI1 regulator. Thus, alterations in SMO protein expression were observed in GBM cells following incubation with different concentrations of FL34 for 72 h, as determined by western blot analysis. However, FL34 did not influence the expression of SMO in U-87 MG or T98G cells [Figure 4]c. Similar results were observed in FL34-treated tumors in which the expression of SMO did not differ significantly from that of the tumors in the control group [Figure 4]d.

Furthermore, crosstalk between HH and the AKT/ERK pathways has been reported.[20] Treatment with FL34 produced various effects on the p-ERK and p-AKT expression levels in GBM cells, as indicated in [Figure 5]a. Lower concentrations of FL34 (0.5 or 5.0 nmol/L in U-87 MG cells and 1.0 or 10.0 nmol/L in T98G cells) did not influence the expression of p-ERK and p-AKT. Treatment with FL34 only marginally decreased the phosphorylation of ERK and AKT at the higher concentration (50.0 nmol/L in U-87 MG cells and 100.0 nmol/L in T98G cells), while no significant alterations in the total ERK and AKT levels were observed in U-87 MG cells [Figure 5]a. In the xenograft model, the expression of p-ERK was decreased compared with that in the control group only in the 30.0 mg/kg FL34-treated group, and there was little difference in the expression of p-AKT between the two FL34-treated groups and the control group. In addition, no significant alterations in the total ERK and AKT levels were observed in vivo in xenograft tumors [Figure 5]b.
Figure 5: Effects of FL34 on biomarkers associated with proliferation, invasion, angiogenesis pathways, and the regulation of apoptosis in U-87 MG and T98G cells and U-87 MG xenograft tumors treated with FL34. Cells were treated with the indicated concentrations of FL34 for 72 h. “Con” refers to untreated control samples. Whole-cell lysates were subjected to western blotting with antibodies against the indicated proteins. Mice bearing U-87 MG tumors were randomized and treated with 15.0 or 30.0 mg/kg FL34 daily for 14 days. Lysates from two or three mice per group were pooled. Each lane represents one protein pool, and two or three pools/groups were subjected to western blot analysis. β-actin was included as a loading control. Representative western blot analysis results are presented for each experiment. (a) Western blot analysis of ERK and AKT in U-87 MG, T98G cells treated with FL34 for 72 h. (b) Western blot analysis of ERK and AKT in U-87 MG xenograft tumors treated with FL34. (c) Western blot analysis of regulators of apoptosis in U-87 MG and T98G cells treated with FL34 for 72 h. (d) Western blot analysis of regulators of apoptosis in U-87 MG xenograft tumors treated with FL34. (e) Western blot analysis of proteins related to invasion and angiogenesis in U-87 MG and T98G cells treated with FL34 for 72 h. (f) Western blot analysis of proteins related to invasion and angiogenesis in U-87 MG xenograft tumors treated with FL34

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To further elucidate the mechanisms underlying the effects of FL34 on GBM cells, the expression of the principal proteins in the apoptotic cascade were examined. Consistent with the increase in the early apoptotic population, a dose-dependent increase in cleaved caspase 3 (c-caspase 3) level in FL34-treated U-87 MG and T98G cells was observed [Figure 5]c. Since Bcl-2 is strongly associated with cell survival [21] and is a downstream factor of the GLI1 oncoprotein, we analyzed Bcl-2 expression by western blotting, observing decreased protein expression in U-87 MG and T98G cells following FL34 treatment for 72 h [Figure 5]c. Moreover, increased protein expression of c-caspase 3 and suppression of Bcl-2 was observed in the tumors treated with 15.0 and 30.0 mg/kg FL34 [Figure 5]d.

The level of FAK activation correlates with metastasis, and HH signaling reportedly plays a critical role in the induction of FAK phosphorylation.[22] Following a 72-h incubation with the aforementioned concentrations of FL34, p-FAK (Tyr397) and FAK expression levels were significantly downregulated in U-87 MG and T98G cells [Figure 5]e. Furthermore, the expressions of MMP9 and MMP2, downregulators of FAK, were also decreased following treatment with FL34 [Figure 5]e. The levels of the FAK and MMP proteins in tumors were also examined by western blotting; as shown in [Figure 5]f, the phosphorylation of FAK, MMP9, and MMP2 was significantly reduced in tumors of the FL34-treated groups compared to those in the control group.

To verify the effect of FL34 on VEGF expression, U-87 MG and T98G cells exposed to FL34 were analyzed for VEGF expression by western blotting. The expression of VEGF was significantly reduced in the FL34-treated GBM cells [Figure 5]e. The inhibitory effect of FL34 on VEGF expression was further confirmed in the U-87 MG tumors by western blotting. Similarly, the expression of VEGF in tumors treated with FL34 was much lower compared to that in the control [Figure 5]f.


 > Discussion Top


The frequent development resistance of cancer cells to SMO inhibitors in SMO-dependent or SMO-independent manners indicates the need for improved therapeutic targets to effectively block the HH signaling pathway. Currently, the antagonists targeting the HH pathway are mainly SMO inhibitors and inhibition of GLI1 is rarely reported.[23]

GLI1 is hyperactivated in a panel of brain tumors including GBM [24] and is the final effector of the HH pathway. Therefore, inhibition of GLI1 may modulate targets downstream of or independently of SMO in HH-dependent tumors. The inhibition of GLI1 may overcome resistance to SMO inhibitors while avoiding adverse effects and may, thus, be a good therapeutic strategy.

This study investigated the effect of FL34 on the growth, angiogenesis, and invasion of GBM cells. Our results demonstrated that the FL34 inhibited the growth of U-87 MG and T98G cells. FL34 also inhibited tumor growth in the U-87 MG xenograft model. FL34 inhibited the transcriptional activity of GLI1 at 19.86 nmol/L, similar to GANT61,[25] resulting in the decreased expression of GLI1 at the mRNA and protein levels in vitro and in vivo in a xenograft model. Simultaneously, the expression of SMO was only marginally influenced by FL34 treatment. These findings suggested that FL34 inhibited GLI1 activation without influencing upstream canonical HH/SMO signaling in vitro and in vivo.

Crosstalk between GLI1 activation and other oncogenic signaling pathways including Ras/MEK/ERK and PI3K/AKT has been reported.[26],[27] Ras/ERK signaling acts upstream of GLI1 through the negative regulator SUFU [28] and ERK is likely to be the principal effector of this pathway. We evaluated the effects of FL34 on the phosphorylation and basal levels of ERK. We found that the FL34-mediated inhibition of GLI1 was not associated with ERK phosphorylation at a low concentration of FL34. In addition, PI3K/AKT inhibitors reportedly confer resistance to SMO agonists through the inactivation of GLI1,[29] supporting the idea that the PI3K/AKT pathway plays an important role in the regulation of GLI1 through crosstalk. Based on our results, the inactivation of GLI1 by FL34 was not due to the inhibition of AKT phosphorylation.

GLI transcription factors stimulate the expression of target genes that directly affect cell proliferation, including Bcl-2.[30] Bcl-2 is a pro-survival protein that is highly expressed in a subset of tumors.[21] Our data suggested that FL34 promoted apoptosis by decreasing the expression of Bcl-2 and increasing the expression of c-caspase 3. Thus, the effect of FL34 on apoptosis might be associated with the inhibition of GLI1.

GBM rarely metastasize out of the central nervous system, and local invasion is the primary means of tumor progression. Hence, more invasive GBM behavior is frequently correlated with a poorer patient prognosis.[31] SHH is positively associated with the phosphorylation of FAK at Tyr397. FAK serves an important role in invasion by regulating the expression of MMP2 and MMP9, which are critical glioma invasion-mediating factors.[32],[33] We demonstrated that FL34 inhibited the invasion of GBM. Furthermore, FL34 suppressed the phosphorylation of FAK and downregulated the expression of MMP2 and MMP9 in vitro and in vivo. These results suggested that the effect of FL34 on invasion might be due to the inhibition of FAK/MMPs, which might be linked to GLI1.

GLI1 regulates the expression of target genes associated with angiogenesis, including VEGF.[34],[35] The results of the present study demonstrated that FL34 inhibited vessel formation in HUVEC-T cells and downregulated VEGF expression in GBM cells and tumors in the xenograft model. Those results suggested that FL34 may exert anti-angiogenic effects through the inhibition of VEGF, which may be regulated by GLI1.

In addition, FL34 could be effective in other brain tumors with high GLI1 expression. The reported downregulation of GLI1 by the GLI1 inhibitor GANT61 or siRNA could increase the sensitivity of glioma cells to TMZ;[36],[37] therefore, FL34 may enhance the anti-tumor effect of TMZ. Their combined use might provide insights into therapeutic strategies for GBM and other types of gliomas.


 > Conclusions Top


The results of this study demonstrated that FL34 was able to inhibit the proliferation and invasion of GBM cells, suppress the formation of blood vessels, and inhibit the growth of GBM in a xenograft model by influencing the expression of Bcl-2, FAK/MMPs, and VEGF and mediated by the inactivation of GLI1 without affecting multiple upstream and cross-linked oncogenic signaling pathways.

Financial support and sponsorship

This study was supported by grants from the CAMS Innovation Fund for Medical Sciences (2016-I2M-1-008).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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