Research ArticleCELLULAR AND MOLECULAR

Mammalian Target of Rapamycin Pathway Promotes Tumor-Induced Angiogenesis in Adenoid Cystic Carcinoma: Its Suppression by Isoliquiritigenin through Dual Activation of c-Jun NH2-Terminal Kinase and Inhibition of Extracellular Signal-Regulated Kinase

Zhi-Jun Sun, Gang Chen, Wei Zhang, Xiang Hu, Cong-Fa Huang, Yu-Fan Wang, Jun Jia and Yi-Fang Zhao
Journal of Pharmacology and Experimental Therapeutics August 2010, 334 (2) 500-512; DOI: https://doi.org/10.1124/jpet.110.167692

Abstract

Tumor-induced angiogenesis is essential for invasive growth and hematogenous metastasis of adenoid cystic carcinoma (ACC), a highly aggressive neoplasm mostly occurring in salivary glands. Previous studies have indicated that strategies directed against angiogenesis will help develop new therapeutic agents for ACC. The Chinese folk medicine licorice has been used for years as a natural remedy for angiogenesis-related diseases. In this study, we examined the effects of isoliquiritigenin (ISL), a flavonoid isolated from licorice, on the growth and viability of ACC cells and observed a concentration-dependent (0–20 μM) inhibition of cell growth without cell death at 24 h. In a further mimic coculture study, ISL effectively suppressed the ability of ACC cells to induce in vitro proliferation, migration, and tube formation of human endothelial hybridoma (EAhy926) cells as well as ex vivo and in vivo angiogenesis, whereas it exerted no effect on EAhy926 cells when added directly or in the presence of vascular endothelial growth factor (VEGF). The data also showed that the specific suppression of tumor angiogenesis by ISL was caused by down-regulation of mammalian target of rapamycin (mTOR) pathway-dependent VEGF production by ACC cells, correlating with concurrent activation of c-Jun NH2-terminal kinase (JNK) and inhibition of extracellular signal-regulated kinase (ERK). Most importantly, ISL also significantly decreased microvessel density within xenograft tumors, associating with the reduction of VEGF production and suppression of the mTOR pathway coregulated by JNK and ERK, as revealed by immunohistochemical studies and clustering analysis. Taken together, our results highlight the fact that ISL is a novel inhibitor of tumor angiogenesis and possesses great therapeutic potential for ACC.

Adenoid cystic carcinoma (ACC) is a glandular epithelial malignancy, which frequently arises in secretary glands, especially the major and minor salivary glands. This neoplasm often presents a prolonged clinical course and is characterized by its infiltrative nature, high incidence of distant metastasis, and poor long-term survival rate (Spiro, 1997). Nevertheless, up to now, there is no effective strategy for the treatment of ACC. Currently, curative surgery followed by postoperative radiotherapy and/or chemotherapy is the preferred therapy for ACC. However, it has been demonstrated to be ineffective for locally recurrent and distantly metastatic ACC. Thus, novel therapeutic approaches to control the development and metastasis of ACC are urgently needed.

Angiogenesis, the formation of new blood vessels from the endothelium of the existing vasculature, has been widely accepted as playing a critical role in tumor progression and metastasis (Folkman, 1990; Hanahan and Folkman, 1996). Avascular tumors can rarely grow beyond 2 to 3 mm in diameter because of the diffusion limit for oxygen and nutrients (Dhanabal et al., 2005). However, once tumors become capable of angiogenesis, they can grow rapidly and metastasize (Lin et al., 2007). Thus, antiangiogenic therapy is regarded as one of the most promising approaches to control tumor growth, invasiveness, and metastasis (De Smet et al., 2006). However, to the best of our knowledge, the significance of antiangiogenic strategies in treatment of ACC, a highly aggressive tumor with exuberant angiogenesis (Zhang et al., 2005; Zhang and Peng, 2009), has not been explored and clarified.

Based on the above concerns, we thought that identification and preclinical/clinical development of novel agents that are nontoxic but can specifically suppress tumor-induced angiogenesis may be a rational approach to develop effective therapies for ACC. Of note, epidemiological studies have indicated that dietary uptake of flavonoids, a large group of polyphenolic compounds present in most fruits, vegetables, and beverages, is associated with a low risk of cancer (Chang et al., 2008). Isoliquiritigenin (ISL), 2′,4′,4′-3-hydroxychalcone (Fig. 1), is one such naturally occurring flavonoid that has been shown to be nontoxic to humans but to have various biological properties, such as antiinflammatory, antioxidant, and antiplatelet aggregation, as well as vasorelaxant and estrogenic effects (Chen et al., 2009). In addition, ISL has also been demonstrated to possess significant antitumor activities, including inhibition of proliferation and/or induction of apoptosis (Jung et al., 2006) as well as prevention of metastasis (Yamazaki et al., 2002). However, there has been no study focusing on the pharmacological activity of ISL in tumor-induced angiogenesis, a frequently mentioned antitumor target for some other flavonoids. Furthermore, it is also worth mentioning that the Chinese folk medicine licorice, from which ISL is derived, has been used for years as a natural remedy for angiogenesis-related diseases. Therefore, in the present study, we explored the effects of ISL on ACC-induced angiogenesis, attempting to address whether the antitumor activities of ISL are due, at least in part, to its suppression of tumor-induced angiogenesis and, more importantly, to develop novel therapeutic agents against ACC.

Fig. 1.
Fig. 1.

Molecular structure of ISL: 2′,4′,4′-3-hydroxychalcone.

Materials and Methods

Materials.

Unless otherwise noted, all chemicals and reagents including ISL were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human VEGF (VEGF165) was from R&D Systems (Minneapolis, MN). Primary antibodies for phospho-Akt (Ser473), Akt, phospho-mTOR (Ser2448), mTOR, phospho-ERK1/2 (Thr-202/Tyr-204), ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-JNK1/2 (Thr183/Tyr185), JNK1/2, phospho-S6 (Ser235/236), S6, phospho-TSC2 (Thr1462), TSC2, phospho-IKK-α/β (Ser176/180), IKK-α/β, phospho-GSK-3β (Ser9), and GSK-3β were purchased from Cell Signaling Technology (Danvers, MA). Primary antibodies against β-actin, mouse CD31 and human CD34, NF-κB p65, and VEGF were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Patients and Immunohistochemistry.

Fifty pathologically confirmed human ACC specimens, with six corresponding pericancerous normal salivary gland (NSG) tissues were collected at the Hospital of Stomatology (Wuhan University, People's Republic of China). All specimens were fixed in 4% buffered paraformaldehyde and embedded in paraffin. The procedures were performed in accordance with the guidelines of the National Institutes of Health regarding the use of human tissues. The study was approved by the review board of the Ethics Committee of the Hospital of Stomatology, Wuhan University. The immunohistochemical analyses were performed according to our previous procedures (Sun et al., 2010). Paraffin-embedded specimens were serially cut into 4-μm sections, deparaffinized, and antigen-retrieved by microwave. After that, the sections were washed with phosphate-buffered saline (PBS) and incubated with 3% hydrogen peroxide and 10% goat or rabbit serum for 15 min, followed by an overnight incubation, either with p-TSC2 (1:200), p-mTOR (1:200), p-S6 (1:200), VEGF (1:100), and CD34 (1:200) antibody. The antibody binding was detected by horseradish peroxidase-conjugated secondary antibody using a diaminobenzidine substrate kit (Dako, Carpinteria, CA) according to the manufacturer's protocol. The negative control slides were obtained by using PBS buffer instead of the primary antibody. Positive controls were oral squamous cell carcinoma slides known to have positive staining for p-mTOR, p-S6, VEGF, and CD34. For each section, at least five fields with typical pathological changes were randomly selected and counted at a magnification of 400 with a light microscope (Leica, Wetzlar, Germany) by two independent observers (Z. J. Sun and G. Chen) blindly. For p-TSC2, p-mTOR, p-S6, and VEGF, the staining scores were evaluated as the summation of staining intensity (0, no staining; 1, mild staining; 2, moderate staining; and 3, intense staining) and the percentage of positive cells (0, <10%; 1, 10–25%; 2, 25–50%; 3, 50–75%; and 4, 75–100% of stained cells) (Zhang et al., 2005). For MVD, the results were calculated according to a modified method based on the technique described by Weidner et al. (1991).

Double-Labeling Immunofluorescence Histochemistry.

Double-labeling immunofluorescence histochemistry analysis was performed on formalin-fixed, paraffin-embedded 5-μm sections. In brief, the tissue sections were dewaxed in xylene, hydrated through graded alcohols and distilled water, and washed thoroughly with PBS. Antigen retrieval was done using 10 mM citrate buffer (pH 6.0) in a microwave oven for 20 min. The sections were allowed to cool down and were rinsed twice with PBS and then incubated in 3% hydrogen peroxide in PBS for 30 min, followed by another incubation in 10% nonimmune donkey serum (Sigma-Aldrich) for 1 h at room temperature. After that, excess solution was discarded, and the sections were incubated with p-S6 (1:100) and VEGF (1:50) antibodies together overnight at 4°C. After sections were washed with PBS, they were sequentially incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody and tetramethylrhodamine B isothiocyanate-conjugated donkey anti-mouse antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA), respectively, for 1 h. Nuclei were counterstained with 4,6-diamidino-2-phenylindole, followed by observation under a fluorescence microscope (Leica).

Cell Culture and Conditional Medium Collection.

The high-metastasis (ACC-M) and low-metastasis (ACC-2) cell lines of human ACC (Guan et al., 1997) and human endothelial hybridoma cell line EAhy926 were obtained from the China Center for Type Culture Collection and were maintained in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

Collection of ACC conditioned medium (CM) was performed as described previously (Ali et al., 2007) with minor modification. In brief, when ACC cells were grown to 70% confluence after overnight incubation, they were starved in serum-deprived DMEM for 12 h to obtain cell synchronization (Zhang and Peng, 2009). Then the serum-deprived medium was discarded, and the cells were exposed to the indicated concentrations of ISL in fresh DMEM containing 10% FBS for 24 h. After that the cells were washed thoroughly with PBS and further incubated in serum-deprived DMEM for another 24 h, and then the cleared supernatants were collected as the CM and stored at −80°C.

Cell Growth Analysis and Cell Viability Measurement.

The growth curves of ACC cells were measured by a CellTiter-Glo (Promega, Madison, WI) luminescent assay. In brief, both ACC-2 and ACC-M cells (5000 cells/well) were seeded in a 96-well microplate (Corning Life Sciences, Lowell, MA) in a final volume of 100 μl. After overnight incubation, the indicated concentrations of ISL in fresh DMEM containing 10% FBS were added and incubated for 24, 48, or 72 h. After completion of the treatment, 100 μl of CellTiter-Glo solution was added to each well and incubated for 20 min at room temperature in the dark. Lysate (50 μl) was transferred to a 96-well white plate (Greiner Bio-One GmbH, Frickenhausen, Germany), and then the luminescence was measured. Percent cell growth was calculated by considering 100% growth at the time of ISL addition.

In the cell viability assays, ACC or EAhy926 cells (3 × 105) were seeded in 6-cm dishes. Experimental conditions were the same as mentioned for the CellTiter-Glo assay, and the cell viability was measured using the Vi-CELL cell viability analyzer (Beckman Coulter, Fullerton, CA). The relative cell viability is presented as a percentage of that of the control group.

Wound-Healing Assay.

EAhy926 cells were seeded in six-well culture plates (Corning Life Sciences) and allowed to grow to 90% confluence, followed by the addition of CM. The center of the cell monolayers was scraped with a sterile micropipette tip to create a gap of constant width. After 12 h, the cells migrated into the gap were counted after fixation and observed under a phase microscope.

Boyden Chamber Migration Assay.

The migration of endothelial cells was also measured in a Transwell Boyden chamber (Corning Life Sciences) containing a 6.5-mm-diameter polycarbonate filter (8-μm pore size). Starved EAhy926 cells (2 × 104 cells/well) were seeded on the upper wells in 100 μl of serum-deprived medium, whereas CM was applied to the lower wells as a chemoattractant. After incubation for 12 h at 37°C, the cells on the upper surface of the membrane were carefully removed with a cotton swab, and migrated cells were fixed with methanol, stained with crystal violet, and then photographed and counted.

Capillary-Like Tube Formation Assay.

A capillary-like tube formation assay was also performed to examine the effect of ISL on ACC-induced angiogenesis in vitro. EAhy926 cells (2 × 105 cell/dish) in CM were seeded into 6-cm culture dishes coated with Matrigel (BD Biosciences, San Jose, CA) and incubated for 24 h at 37°C. After that, the cells were fixed and stained with acridine orange. The formation of capillary-like structures was captured, and the tubes were scanned and quantitated using Image-Pro Plus (Media Cybernetics, Bethesda, MD).

Rat Aortic Ring Assay.

The thoracic aortas were harvested from Sprague-Dawley rats (6 weeks of age), placed in the 48-well plates coated with 120 μl of Matrigel (BD Biosciences), and sealed in place with an overlay of 50 μl of Matrigel. Conditioned medium treated with or without ISL was added to the wells in a final volume of 200 μl. As controls, medium alone was assayed. On day 6, the microvessel outgrowth was photographed under a phase microscope. The assay was scored from 0 (least positive) to 5 (most positive) in a double-blinded manner.

Chicken Chorioallantoic Membrane Assay.

A modified chick chorioallantoic membrane (CAM) assay was performed as described previously (Kim et al., 2002). In brief, the sterile filter paper discs (8 mm) containing 20 μl of CM with or without ISL treatment were placed on the CAM of a 9-day-old embryo. After a 72-h incubation, the area around the discs was photographed with a stereo microscope, and the number of newly formed vessels was counted by two observers in a double-blind manner. Each assay takes 8 to 10 eggs.

Matrigel Plug Assay.

An in vivo Matrigel plug assay was performed as described previously (Passaniti, 1992). In brief, 0.6 ml of Matrigel containing 0.2 ml of CM was injected subcutaneously into nude mice. After 7 days, the skin of the mouse was pulled back to expose the Matrigel plug completely. The plugs were photographed and then hemoglobin was measured using the Drabkin reagent kit 525 (Sigma-Aldrich) for the quantification of blood vessel formation.

Semiquantitative RT-PCR.

To evaluate the mRNA expression of growth factors in ACC cells after ISL treatment, semiquantitative RT-PCR was performed. In brief, ACC-M cells were exposed to the indicated concentrations of ISL in DMEM containing 10% FBS for 24 h, and then total RNA from the cells was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA). Aliquots (1 μg) of RNA were reverse transcribed to cDNA (20 μl) with oligo(dT) and avian myeloblastosis virus reverse transcriptase (Takara, Kyoto, Japan). One fifth of the cDNA was used as a template for PCR using a PE9700 RT-PCR system (Applied Biosystems, Foster City, CA). The primer sequences for PCR were designed as follows: VEGF, 5′-TCATCTCTCCTATGTGCTGGC-3′ and 5′-ATGAACTTTCTGCTCTCTGG-3′; basic fibroblast growth factor (bFGF), 5′-GTGTGTGCTAACCGTTACCT-3′ and 5′-GCTCTTAGCAGACATTGGAAG-3′; granulocyte colony-stimulating factor (G-CSF), 5′-AGACAGGGAAGAGCAGAACGG-3′ and 5′-GCCAGAGTGAGGGGTGCAA-3′; platelet-derived growth factor (PDGF), 5′-CCCCTGCCCATTCGGAGGAAGAG-3′ and 5′-TTGGCCACCTTGACGCTGCGGTG-3′; and glyceraldehyde-3-phosphate dehydrogenase (control), 5′-AACGGATTTGGTCGTATTGGG-3′ and 5′-CAGGGGTGCTAAGCAGTTGG-3′. The PCR products were electrophoresed in 2% agarose gel and visualized with ethidium bromide. The intensity of each band was analyzed densitometrically using GeneTools software (Syngene, Cambridge, UK).

ELISAs for Secretion of Growth Factors.

ACC-M cells were starved in serum-deprived DMEM for 12 h to obtain cell synchronization. Then the serum-deprived medium was discarded, and the cells were exposed to the indicated concentrations of ISL in fresh DMEM containing 10% FBS for 24 h. After that, the cells were washed thoroughly with PBS and further incubated in serum-deprived DMEM for another 24 h to allow angiogenic factor production. Subsequently, the culture medium was collected and used to determine the secretion of VEGF, bFGF, G-CSF, and PDGF using commercially available kits (R&D Systems) according to the manufacturers' recommendations.

Western Blot Analysis.

ACC-M cells were treated with the indicated concentrations of ISL in DMEM containing 10% FBS for 24 h. Then the cells were lysed, and the total protein was separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA). The immunoblots were cultured overnight at 4°C with the corresponding primary antibodies in blocking solution, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Pierce Chemical, Rockford, IL). Then blots were developed by a Super Enhanced chemiluminescence detection kit (Applygen Technologies Inc., Beijing, People's Republic of China). β-Actin was detected on the same membrane and used as a loading control.

Nude Mice Xenografts.

Female BALB/c nude mice (18–20 g; 6–8 weeks of age) were purchased from the Experimental Animal Center of Wuhan University in pressurized ventilated cages according to institutional regulations. All studies were approved and overseen by the local institutional animal care and use committee. ACC-M cells (2 × 106 in 0.2 ml of medium) were collected from subconfluent cultures and inoculated subcutaneously into the flank of the mice. After 7 days, tumor-bearing mice were randomly divided into three groups, which were, respectively, given corn oil (control, 100 μl p.o. daily; n = 8), a low dose of ISL (0.5 g/kg p.o. daily; n = 8), or a high dose of ISL (1 g/kg p.o. daily; n = 8) for 30 consecutive days. Tumor growth was determined by measuring the size of the tumors daily for 30 days. Tumor volumes were calculated according to the formula (width2 × length)/2. The mice were euthanized at day 30, and the tumors were captured, photographed, and then embedded in paraffin for MVD detection and immunohistochemical analysis.

Microvessel Density in Xenograft Tumor Samples.

To quantify angiogenesis in xenograft tumor samples, microvessels were identified by CD31 immunofluorescence staining. The CD31-stained sections were observed under a Leica fluorescence microscope, and images were captured from different areas in each section. Then, the images were processed using Image-Pro Plus, and the vessel density was estimated by measuring the pixel intensity in each field of view. The vessel density of each group is presented as intensity per pixel. A total of 20 high-power fields were examined from three tumors of each of the treatment groups.

Hierarchical Clustering, Data Visualization, and Statistical Analysis.

The staining scores that resulted from immunohistochemical analyses of xenograft tumor samples were converted into scaled values centered on zero in Microsoft Excel. The hierarchical analysis was done using Cluster 3.0 (http://bonsai.ims.u-tokyo.ac.jp/∼mdehoon/software/cluster/) with average linkage based on Pearson's correlation coefficient as the selection variable on the unsupervised approach (Eisen et al., 1998). The results were visualized using Java TreeView 1.0.5 (http://jtreeview.sourceforge.net/) as described previously (Saldanha, 2004). The clustered data were arranged with markers on the horizontal axis and tissue samples on the vertical axis. Two biomarkers with a close relationship are located next to each other.

All data are expressed as means ± S.E.M. of three independent experiments. One-way analysis of variance, Student-Newman-Keuls test, and Spearman rank correlation test were used for statistical analysis. P < 0.05 was considered significantly different.

Results

ISL Inhibits the Growth of ACC Cells and Prevents ACC-Induced Proliferation of EAhy926 Cells.

In this study, we initially determined the growth characteristics of both ACC-M and ACC-2 cells in response to ISL treatment using the CellTiter-Glo assay. As shown in Fig. 2, A and B, ISL (0–20 μM) concentration- and time-dependently decreased the growth activity of both ACC-M and ACC-2 cells, but this effect was more prominent in the ACC-M cell line. Moreover, results from the cell viability assays (Fig. 2, C and D) revealed that treatment with increased concentrations of ISL (5, 10, and 20 μM) at 24 h did not affect the viability of either ACC-M or ACC-2 cells, indicating that the growth inhibitory effect of ISL at 24 h was not associated with the cytotoxicity. In contrast, a significant decrease in ACC cell viability was noticed after treatment with higher concentrations of ISL (10 and 20 μM) for 48 and 72 h. Thus, the 24-h treatment period of ISL (0, 5, 10, and 20 μM) was selected as the nontoxic treatment condition used in the CM collection and the following studies.

Fig. 2.
Fig. 2.

Inhibitory effect of ISL on ACC cell growth and viability and on ACC-induced proliferation of EAhy926 cells. ACC-M (A) and ACC-2 (B) cell growth was measured by the CellTiter-Glo assay after treatment with the indicated concentrations of ISL in DMEM containing 10% FBS for 24, 48, and 72 h. Results are presented as a percentage at the time of ISL addition. ACC-M (C) and ACC-2 (D) cell viability was measured using the Vi-CELL cell viability analyzer after treatment with the indicated concentrations of ISL in DMEM containing 10% FBS for 24, 48, and 72 h. Results are presented as a percentage of the control group. EAhy926 cell viability after treatment with DMSO (vehicle) alone, ISL alone, VEGF alone, and VEGF plus ISL, as well as CM from ACC-M (E) or ACC-2 (F) cells pretreated with or without ISL was measured using the Vi-CELL cell viability analyzer at 24 h. Results are presented as a percentage of the vehicle-treated control group. All data are presented as means ± S.E.M. from three different experiments performed in duplicate. ∗, P < 0.05; ∗∗, P < 0.01 versus the vehicle-treated group; †, P < 0.05; ††, P < 0.01 versus the CM without ISL treatment group.

Angiogenesis is a complex biological process that requires precise coordination of multiple and various steps, including endothelial cell proliferation, migration, and tube formation (Folkman, 1995; Bussolino et al., 1997). To ascertain the inhibitory effect of ISL on tumor-induced angiogenic features of endothelial cells, a set of angiogenesis assays in vitro was performed. First, we explored the effect of ISL on ACC-induced EAhy926 cell proliferation. The number of EAhy926 cells was measured in the absence or presence of CM with or without the indicated concentrations of ISL. As shown in Fig. 2, E and F, CM without ISL treatment significantly increased viable EAhy926 cell numbers. However, pretreatment with ISL effectively blocked CM-induced proliferation of EAhy926 cells in a concentration-dependent manner. Furthermore, we also found that this preventive effect of ISL is more prominent in ACC-M cell line, consistent with the results from cell growth analyses. We next tried to explore the antiproliferation effect of ISL on untreated and VEGF-treated EAhy926 cells. However, treatment with ISL showed no influence on the proliferation of untreated or VEGF-treated EAhy926 cells. These findings initially indicated that ISL may only selectively target the tumor-induced angiogenic process.

ISL Inhibits ACC-Induced Endothelial Cell Migration and Tube Formation.

Because cell migration is necessary for the angiogenic process of endothelial cells, we then investigated the effect of ISL on ACC-induced EAhy926 cell migration. Results from the wound healing assays revealed that ISL concentration-dependently prevented CM-induced mobility of EAhy926 cells (Fig. 3A). In addition, the Boyden chamber assay also confirmed the preventive effects on ACC CM-induced EAhy926 cell migration (Fig. 3A).

Fig. 3.
Fig. 3.

ISL prevents ACC-induced angiogenesis in vitro, ex vivo, and in vivo. A, the wound-healing assay and Transwell Boyden chamber assay, as well as capillary-like tube formation assay were performed to measure in vitro effects of ISL on the ACC-induced angiogenic features of EAhy926 cells. Values are presented as a percentage of the vehicle-treated control group in the ACC-2 cell line. B, effects of ISL on the angiogenic features of untreated and VEGF-treated EAhy926 cells. C, effect of ISL on ACC-induced vessel sprouting in the rat aortic ring assay ex vivo. D, effect of ISL on ACC-induced angiogenesis in the CAM assay in vivo. E, effect of ISL on ACC-induced vessel formation in the Matrigel plug assay in vivo. mRNA expression (F) and protein secretion (G) levels of VEGF were determined by RT-PCR and ELISAs, respectively, after incubation with indicated concentrations of ISL for 24 h. All data are presented as means ± S.E.M. from three different experiments performed in duplicate. ∗, P < 0.05; ∗∗, P < 0.01 versus the vehicle-treated group; †, P < 0.05; ††, P < 0.01 versus the CM without ISL treatment group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

It is also well known that endothelial cells can form capillary-like structures spontaneously on Matrigel, which is crucial in vasculogenesis. Therefore, we further determined the effect of ISL on ACC-induced tube formation of EAhy926 cells. CM pretreated with or without the indicated concentrations of ISL was added to the Matrigel-coated dishes seeded with EAhy926 cells. The total number of branched tubes per field was counted after a 24-h incubation. As shown in Fig. 3A, CM without ISL treatment significantly promoted the capillary structure formation of EAhy926 cells compared with the negative controls. However, the induced tube formation was effectively prevented by pretreatment with ISL in a concentration-dependent manner.

In addition, our results also revealed that ISL failed to affect the migration and tube formation of untreated or VEGF-treated EAhy926 cells, repeatedly demonstrating that ISL only selectively suppressed the tumor-induced angiogenic process but showed no influence on preexisting normal angiogenesis (Fig. 3B). In addition, all of the above results revealed that the tumor angiogenesis prevention activity exerted by ISL in vitro is more prominent in the high-metastasis cell line ACC-M compared with that in the low-metastasis cell line ACC-2, suggesting high specificity. Thus, the ACC-M cell line was chosen for the subsequent studies.

ISL Prevents ACC-Induced Angiogenesis Ex Vivo and In Vivo.

To verify the preventive effect of ISL on ACC-induced angiogenesis in vitro, a further series of ex vivo and in vivo assays was carried out. First, we performed ex vivo aortic ring assays using isolated aortas from mice. The 1- to 1.5-mm-long aortic rings were put on Matrigel and covered by another Matrigel layer, followed by addition of ACC-M CM with or without ISL pretreatment. After incubation for 6 days, the microvessel outgrowth from the aortic rings was analyzed. As shown in Fig. 3C, the results demonstrated that ISL at 20 μM nearly blocked all of the new microvessel outgrowth induced by CM from ACC-M cells.

The ability of ISL to prevent in vivo ACC-induced angiogenesis was next examined using the CAM assay and Matrigel plug assay. In the CAM assays, paper discs were prepared and inserted into ACC-M CM treated with or without ISL and then placed on the surface of a CAM. At the end of treatment, the newly formed microvessels around the loaded disk were counted and analyzed. As shown in Fig. 3D, treatment with 20 μM ISL almost reversed the stimulated effect of ACC-M CM on new microvessel formation. Furthermore, results from the Matrigel plug assays (Fig. 3E) also confirmed this conclusion in that plugs with ACC CM without ISL treatment appeared to be dark red in color, but plugs with Matrigel alone or with CM with 20 μM ISL treatment were pale. The hemoglobin content within the Matrigel plugs was measured to quantify the tumor-induced angiogenesis prevention effect of ISL, and the results are shown in Fig. 3E. Taken together, our data strongly indicated, for the first time, that ISL is a potent and specific inhibitor of tumor-induced angiogenesis in ACC.

ISL Down-Regulates VEGF Production in ACC Cells.

Angiogenesis is a complex biological process involving a wide variety of regulators. The above results have indicated that ISL possesses a high potential to prevent ACC-induced angiogenesis both in vitro and in vivo. To further elucidate the mechanisms underlying this preventive effect of ISL, the mRNA expression and protein secretion levels of some key angiogenic factors were tested by semiquantitative RT-PCR and ELISAs, respectively. As shown in Fig. 3, F and G, ISL treatment significantly down-regulated VEGF production in both mRNA expression and protein secretion levels. However, the production of bFGF, G-CSF, or PDGF was not altered (data not shown). Indeed, VEGF is generally considered to be the most potent and specific angiogenic factor that is closely associated with the angiogenic process in ACC. Thus, it is rational to conclude that ISL down-regulated the VEGF production by ACC cells and led to the prevention of angiogenesis induction.

ISL Inactivates the mTOR Signaling Pathway Coregulated by JNK and ERK.

To unmask the precise molecular mechanisms underlying the above-demonstrated inhibitory activities of ISL, a set of Western blot analyses was performed. The data revealed that ISL significantly suppressed the phosphorylation of mTOR, S6, and ERK and also up-regulated the phosphorylation level of TSC-2 and JNK (Fig. 4A) in a concentration- and time-dependent manner, but it failed to affect the activation status of PI3K, Akt, and p38 or of NF-κB and IκBα kinase (IKK) (data not shown). To further determine the functional significance of mTOR suppression in the inhibitory activities of ISL, the specific inhibitor of mTOR, rapamycin, was given to ACC-M cells before treatment with ISL. Rapamycin (100 nM) significantly suppressed the activity of mTOR as well as that of its downstream target S6 (Fig. 4B), consistent with the nearly complete blockade of ISL-mediated down-regulation of VEGF production (Fig. 4C) and prevention of ACC-induced tube formation of endothelial cells (Fig. 4D). However, treatment with rapamycin showed no influence on ISL-mediated alteration of TSC, ERK, or JNK (Fig. 4B). These results indicate that the mTOR signaling pathway may be a crucial downstream mechanism implicated in the inhibitory activities of ISL in ACC.

Fig. 4.
Fig. 4.

ISL suppresses the mTOR signaling pathway through dual JNK activation and ERK inactivation. A, ACC-M cells were treated with the indicated concentrations of ISL in DMEM containing 10% FBS for indicated time. The expression levels of p-ERK1/2 (Thr-202/Thr-204), p-JNK1/2 (Thr-183/Thr-185), p-TSC2 (Thr-1462), p-mTOR (Ser-2448), and p-S6 (Ser-235/236) and their corresponding total protein were determined by Western blot analysis. B, ACC-M cells were pretreated with SP600125, PD98059, or rapamycin for 1 h, followed by incubation with ISL for 24 h. The expression levels of p-ERK1/2, p-JNK1/2, p-TSC2, and p-S6 were analyzed by Western blotting. C and D, ACC-M cells were exposed to SP600125, PD98059, or rapamycin, respectively, for 1 h, followed by incubation with ISL in DMEM containing 10% FBS for 24 h. Then the cells were washed thoroughly with PBS and further incubated in serum-deprived DMEM for another 24 h to allow angiogenic factor production. After that the cleared supernatants were collected as the CM. The protein secretion level of VEGF in the CM was quantified using an ELISA. Percent VEGF production was calculated by considering 100% production together at the time of ISL addition. When CM was added to EAhy926 cells, the angiogenic activity was measured by the capillary-like tube formation assay (D). Percent CM-induced tube formation was calculated by considering 100% formation together at the time of ISL addition. All data are presented as means ± S.E.M. from three different experiments performed in duplicate. ∗, P < 0.01 versus the corresponding group without ISL treatment; †, P < 0.01 versus the control group with only ISL treatment but without any other pretreatment.

To further elucidate the relationship between ISL-induced JNK activation and ERK inactivation, as well as their relevance with the TSC/mTOR/S6 pathway, the specific pharmacological inhibitors of both JNK [anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125)] and ERK [2′-amino-3′-methoxyflavone (PD98059)] were used. Pretreatment with SP600125 significantly but partially enhanced the preventive effect of ISL on VEGF production (Fig. 4C) and the ACC-induced angiogenic process (Fig. 4D). Furthermore, SP600125 pretreatment also significantly enhanced ISL-mediated TSC2 activation and mTOR/S6 suppression, but not ISL-mediated inhibition of ERK (Fig. 4B). On the other hand, PD98059 partially but significantly abrogated the preventive effect of ISL on VEGF production as well as ACC-induced tube formation of endothelial cells (Fig. 4, C and D), and concurrently blocked ISL-mediated TSC2 activation and mTOR/S6 suppression, but not ISL-mediated activation of JNK (Fig. 4B). These data indicate that JNK and ERK might be concurrently but independently involved in ISL-mediated suppression of the TSC2/mTOR/S6 pathway and ACC-induced angiogenesis. Nevertheless, the precise mechanisms by which JNK and ERK regulate the TSC2/mTOR/S6 pathway in ACC still needs further investigation.

ISL Suppresses Tumor Angiogenesis and Prevents Tumor Growth In Vivo.

To further authenticate the angiogenesis prevention and growth inhibition ability of ISL, ACC-M xenografts in nude mice were established. The results showed that the growth of ACC-M tumors was significantly inhibited by ISL treatment in a dose-dependent manner compared with the vehicle controls (Fig. 5, A and B), whereas no toxicity was observed (Fig. 5C). In a similar manner, down-regulated proliferation activity revealed by positive Ki67 expression, as well as reduced MVD marked as CD31-positive staining, was also observed in high-dose ISL-treated ACC-M tumors (Fig. 5D). To further correlate these in vivo tumor-therapeutic effects of ISL to the mechanisms identified in vitro, we evaluated the activation status of some key signaling pathways in ISL-treated ACC-M tumor tissues by immunohistochemical analyses. As the results show, a decrease in S6 phosphorylation in ISL-treated tumors clearly contrasted with the high activation level of S6 in the control group (Fig. 5D), reflecting the hyperactivity of the mTOR signaling pathway in ACC. Concomitant with the reduction in S6 phosphorylation, we also noted a significant decrease in VEGF production in ISL-treated tumors (Fig. 5D). More importantly, cluster analysis of the immunohistochemical results indicated the therapeutic significance of ISL treatment. As shown in Fig. 5E, all control samples clustered together, and tissues treated with a low dose of ISL (0.5 g/kg) were clustered together and adjacent to the controls. Nearly all samples treated with a high dose of ISL (1 g/kg) were closely clustered. From the heat map approach, it was also clear that Ki67, p-mTOR, p-S6, and VEGF clustered together, and p-JNK as well as p-ERK were clustered closely to them, but p-PI3K, p-Akt, and p-p38 were distantly clustered. All of the above results are consistent with our in vitro findings and repeatedly demonstrated that ISL significantly and specifically suppressed tumor-induced angiogenesis in ACC by inhibiting the mTOR signaling pathway through dual JNK activation and ERK inactivation.

Fig. 5.
Fig. 5.

ISL prevents tumor angiogenesis and growth in vivo. A, tumor regression observed in ACC xenografts treated with ISL. ACC-M cells were used to establish xenografts in athymic nu/nu mice, and tumor-bearing animals were given ISL (0.5 or 1 g/kg p.o. daily; n = 8) or corn oil (vehicle, 100 μl p.o. daily; n = 6) for 30 days. An example of tumor regression in high-dose (1 g/kg) ISL-treated animals is depicted. B, tumor size from ACC-M xenografts in both ISL- and vehicle-treated groups was assessed daily, as indicated. All values are presented as means ± S.E.M. (n = 6 or 8). C, body weight of mice was measured daily and indicated no side effect of ISL treatment. All values are presented as means ± S.E.M. (n = 6 or 8). D, immunohistochemical analysis of the indicated biomarkers in both control and ISL-treated ACC-M tumor tissues. E, hierarchical clustering of immunohistochemical results from the xenograft tumors after treatment with vehicle control (C1–5), low-dose ISL (ISL-L1–8), and high dose ISL (ISL-H1–8). Light yellow, high staining scores; light blue, low staining scores.

Immunohistochemical Analyses of Clinical Specimens Confirmed the High Activation Status of the TSC2/mTOR/S6 Pathway and Its Essential Role in Angiogenesis in ACC.

To make our above findings more clinically significant and therapeutically meaningful, we further evaluated the natural activation status of the mTOR pathway in ACC and attempted to explore its relevance with angiogenesis. We initially tested and compared the activation status of the mTOR signaling pathway in two ACC cell lines. The results revealed that the activation status of the mTOR pathway in the high-metastasis cell line ACC-M was significantly higher than that in the low-metastasis cell line ACC-2 (Fig. 6A), correlating with the relatively higher VEGF production level (Fig. 6, B and C) and the angiogenesis-induction ability (Fig. 6D). More importantly, treatment with rapamycin significantly down-regulated VEGF production and obviously prevented the angiogenic process of endothelial cells induced in both ACC-M and ACC-2 cells but again was more prominent in ACC-M cells (Fig. 6D). The above data indicated that the activation of the mTOR pathway in ACC cells might contribute greatly to the angiogenesis-induction potential. To further confirm the above findings in ACC cell cultures, we then assessed the natural activation status of the mTOR pathway in ACC tissue specimens and explored its relevance with angiogenesis in the tumors. Representative examples of immunohistochemical results are shown in Fig. 7A. In the selected ACC cases, it was clear that p-S6 was intensely stained in the cytoplasm of most ACC cells accompanied by strong staining of p-mTOR in the cytoplasm and/or nuclei. In contrast, the staining of p-TSC2 was almost not detectable. Furthermore, positive staining of VEGF was obviously detected in the same area. In addition, the double-labeling immunofluorescence histochemistry of p-S6 and VEGF further confirmed the concurrence of S6 activation and VEGF production (Fig. 7B) in ACC tissues. More importantly, the Spearman rank test showed significant correlation among the staining level of p-TSC2, p-mTOR, p-S6, and VEGF, as well as MVD (Table 1), strongly indicating that the high activation status of mTOR in ACC might have greatly contributed to the exuberant angiogenesis involving the regulation of VEGF production. Taken together, the findings in the ACC cell cultures as well as in the ACC tissue specimens were highly consistent, which together revealed that the mTOR signaling pathway is ubiquitously activated and correlated with the exuberant angiogenesis in ACC and more importantly demonstrated the high therapeutic potential of mTOR inhibition.

Fig. 6.
Fig. 6.

Activation status of the mTOR signaling pathway in both ACC-M and ACC-2 cell lines, as well as its relevance in angiogenesis induction ability. A, expression levels of p-TSC2, p-mTOR, and p-S6 and their corresponding total protein were determined by Western blot analysis in ACC-M and ACC-2 cell lines, respectively. The mRNA expression (B) and protein secretion (C) of VEGF in both ACC-M and ACC-2 cell lines, treated with or without rapamycin, were measured by the RT-PCR and ELISA, respectively. D, the cell migration assay and capillary-like tube formation assay were performed to detect and compare the angiogenesis induction ability of both ACC-M and ACC-2 cells, as well as their response to rapamycin (Rap) treatment. The results are presented as a percentage of the control group. All data are presented as means ± S.E.M. from three different experiments performed in duplicate. Values without a common letter differ, P < 0.05.

Fig. 7.
Fig. 7.

Concurrent mTOR pathway activation and VEGF production in ACC. A, immunohistochemical staining of p-TSC2, p-mTOR, p-S6, and VEGF in both NSG and ACC tissues. B, double-labeling immunofluorescence histochemistry of p-S6 and VEGF in both NSG and ACC tissues. FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine B isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole.

View this table:
TABLE 1

Spearman rank test analyses of immunostaining

Values represent number of cases analyzed (P).

Discussion

In the present study, ISL, a flavonoid isolated from licorice, was revealed to be a potent therapeutic agent for ACC. Although this natural flavonoid has been extensively considered as an antineoplastic agent in various human cancers (Hsu et al., 2005; Yoshida et al., 2008; Ye et al., 2009) and has shown inhibitory effects on the phorbol myristate acetate-induced angiogenic process of endothelial cells (Kang et al., 2010), this is the first study regarding prevention of tumor angiogenesis as an important component of the antitumor mechanisms of ISL. In this study, we demonstrated for the first time that ISL was a potent inhibitor of tumor-induced angiogenesis in ACC. Initially, by using growth analysis and viability measurement, we determined that ISL at a concentration of 0 to 20 μM did not induce significant ACC cell death but only effectively decreased the cell growth activity. Further investigation revealed that ISL at these concentrations not only significantly prevented ACC-mediated proliferation, migration, and tube formation of the human endothelial hybridoma (EAhy926) cell line in vitro but also suppressed the ACC-induced angiogenic process ex vivo and in vivo. More importantly, ISL obviously decreased the MVD within xenograft tumors, concomitant with the down-regulated proliferative activity of ACC cells. Taken together, the results suggest that ISL possesses great therapeutic potential for ACC because of its function in preventing tumor-induced angiogenesis.

Previous studies have implicated multiple growth factors, such as VEGF, bFGF, G-CSF, and PDGF, in tumor growth, angiogenesis, and metastasis (Slomiany and Rosenzweig, 2006; Passam et al., 2008). Among them, VEGF is generally considered to be the most potent and specific angiogenic factor that is closely associated with aggressive human cancer cells, including ACC (Zhang et al., 2005; Zhang and Peng, 2009). The present study revealed that exposure of ACC cells to ISL resulted in significant down-regulation of VEGF production. In contrast, the production of bFGF, G-CSF, or PDGF was unaffected. Taken together, these results suggest that ISL-mediated suppression of tumor angiogenesis in ACC is most likely caused by VEGF down-regulation.

Recent research has indicated that NF-κB is tightly involved in the production of VEGF in different cell lines (Josko and Mazurek, 2004; Korkolopoulou et al., 2008; Zhang and Peng, 2009), including ACC cells (Zhang et al., 2005; Zhang and Peng, 2009). Furthermore, several flavonoids including ISL have been reported to inhibit NF-κB activation (Kwon et al., 2007). Thus, it is reasonable to hypothesize that ISL may down-regulate VEGF production and prevent ACC cell-induced angiogenesis through inhibition of NF-κB activation. However, unexpectedly, our data showed that ISL failed to inactivate NF-κB or its upstream regulator IKK. Thus, we explored other possible intracellular signals targeted by ISL, in particular, those with an important role in tumor angiogenesis. It is noteworthy that the TSC/mTOR signaling axis is one such pathway that has been extensively implicated in the angiogenic process during the malignant progression of cancers (Lee et al., 2007). The TSC comprises two subunits, the tumor suppressor genes TSC1 and TSC2. TSC1 stabilizes TSC2 through binding with it, thereby preventing TSC2 from ubiquitination and degradation (Benvenuto et al., 2000). TSC2 acts as a GTPase-activating protein to regulate RHEB function through converting RHEB from an active GTP-bound form to an inactive GDP-bound form (Benvenuto et al., 2000). Inactivation of TSC2 represses GTPase-activating protein activity and allows GTP-bound RHEB to accumulate. GTP-bound RHEB is known to activate mTOR, a highly conserved serine/threonine kinase, which in turn phosphorylates and activates S6K, leading to activation of the downstream mTOR/S6 pathway. In contrast, active TSC2 reduces GTP-bound RHEB accumulation and prevents mTOR and S6 activation. More importantly, activation of the TSC/mTOR signaling pathway has been demonstrated to cause up-regulation of VEGF through increases of both transcription and translation (Klos et al., 2006), leading to promotion of angiogenesis, enhanced growth, and ultimately to tumorigenesis (Inoki et al., 2005). Indeed, the ubiquitous activation of the mTOR pathway, resulting from TSC inactivation, has been revealed to closely associate with the extensive abnormal vasculization in various tumors (Onda et al., 1999; Kwiatkowski et al., 2002; Lee et al., 2007), and strategies specifically against mTOR may mitigate tumor progression due to the prevention of angiogenesis (Lee et al., 2007). In the present study, we noted that the activity of mTOR as well as that of its major downstream target S6 was significantly down-regulated by ISL, and the phosphorylation level of TSC2 was obviously up-regulated, demonstrating probable involvement of the TSC2/mTOR/S6 pathway.

To make the above findings more clinically significant and therapeutically meaningful, we explored the natural activation status of the mTOR pathway as well as its relevance for angiogenesis in ACC. Immunohistochemical analyses showed that the phosphorylation status of mTOR and S6 is significantly higher in ACC tissues than in NSG tissues, correlating with decreased TSC2 activation, up-regulated VEGF production, and increased MVD. Furthermore, the double-labeling immunofluorescence histochemistry analysis also confirmed the concurrence of S6 activation and VEGF production in ACC tissues. More importantly, the phosphorylation levels of TSC2, mTOR, and S6 are significantly correlated with each other and also with VEGF production as well as MVD, as demonstrated by the Spearman rank test. Of interest, our findings also revealed that the activation status of the TSC/mTOR/S6 signaling pathway in the high-metastasis cell line ACC-M was significantly higher than that in the low-metastasis cell line ACC-2, correlating with the relatively higher VEGF production level and angiogenesis induction ability. Taken together, the data strongly indicate that activation of the mTOR signaling pathway is essential for the angiogenic process during the malignant progression of ACC. To the best of our knowledge, this is the first time that the mTOR pathway was reported to be ubiquitously activated in ACC, revealing in particular its potential role in promotion of angiogenesis. These findings may not only lead to better understanding of the molecular pathogenesis of ACC but also provide a basis for a rational approach to development of molecular targeted therapies against this aggressive tumor, just like the ISL treatment reported here. To further validate the above conclusion and confirm the potential of mTOR inhibition in antiangiogenic therapies against ACC, we then tested the effect of rapamycin, a specific mTOR inhibitor, on VEGF production and induction of angiogenesis by ACC cells. As expected, rapamycin significantly down-regulated VEGF production and prevented the angiogenic process of endothelial cells induced in both ACC-M and ACC-2 cells, but again more prominent in ACC-M cells. Taking the above results together, we may conclude that ISL-mediated suppression of tumor angiogenesis in ACC is due, at least in part, to its down-regulation of the TSC2/mTOR/S6 signaling pathway, which was ubiquitously activated and correlated with the exuberant angiogenesis in ACC.

Serine-threonine kinase Akt (protein kinase B), the direct downstream effector of PI3K, is the most frequently mentioned upstream regulator of the TSC/mTOR signaling pathway. However, during our attempt to understand the upstream molecular mechanisms for ISL-induced mTOR inactivation, we unexpectedly found that the activation status of the PI3K/Akt pathway was not altered by ISL. Because other upstream signals including IKKβ, GSK, and MAPKs could also regulate mTOR activity through Akt-independent pathways (Ma et al., 2005, 2007; Lee et al., 2007; Buller et al., 2008), we examined whether these molecules play roles in ISL-mediated mTOR inhibition. The results show that ISL treatment concurrently activated JNK and inactivated ERK in ACC cells but had no effect on GSK or p38 MAPK, another subfamily of MAPKs. In an attempt to gain more insights into the underlying mechanisms by using the specific inhibitors of mTOR (rapamycin), JNK (SP600125), and ERK (PD98059), we determined that ISL concurrently but independently activated JNK and inactivated ERK, respectively, to up-regulate TSC2 activity, resulting in mTOR inhibition in ACC cells. Most importantly, the in vivo studies further verified the in vitro findings. ISL treatment effectively reduced MVD and prevented tumor growth of ACC-M cells in the nude mouse xenograft model. Furthermore, immunohistochemical analyses showed that ISL significantly blocked the activation of the mTOR pathway and down-regulated the production of VEGF. Furthermore, the cluster analysis revealed that the inactivated mTOR pathway and down-regulated VEGF production were closely related to each other and to JNK activation and ERK inactivation but showed no correlation with the PI3K/Akt or p38 MAPK signaling pathway. All of the above results are consistent with our in vitro findings.

In conclusion, the present study demonstrated for the first time that ISL, a natural flavonoid derived from licorice, significantly and specifically suppressed tumor-induced angiogenesis in ACC both in vitro and in vivo, correlating with inhibition of the mTOR signaling pathway through dual JNK activation and ERK inactivation. Most importantly, we revealed, also for the first time, that the mTOR signaling pathway was indeed ubiquitously activated and correlated with the exuberant angiogenesis in ACC. Our findings systematically dissected the effects of ISL on the mTOR signaling pathway in ACC, determined the importance of mTOR inhibition for the antiangiogenic activity of ISL, and shed new light on the mechanisms of anticancer activities of ISL, as well as indicating its great therapeutic potential for ACC.

Footnotes

  • This study was supported in part by the National Natural Science Foundation of China [Grants 30872894, 30973330, 30801305] (to Y.F.Z. and J.J.); and the National Undergraduate Innovative Experiment Project [Grant 091048654] (to G.C.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.110.167692.

  • ABBREVIATIONS:

    ACC
    adenoid cystic carcinoma
    ISL
    isoliquiritigenin
    VEGF
    vascular endothelial growth factor
    mTOR
    mammalian target of rapamycin
    ERK
    extracellular signal-regulated kinase
    MAPK
    mitogen-activated protein kinase
    JNK
    c-Jun NH2-terminal kinase
    NSG
    normal salivary gland
    TSC
    tuberous sclerosis complex
    NF-κB
    nuclear factor-κB
    PBS
    phosphate-buffered saline
    p
    phospho
    MVD
    microvessel density
    DMEM
    Dulbecco's modified Eagle's medium
    FBS
    fetal bovine serum
    CM
    conditioned medium
    CAM
    chick chorioallantoic membrane
    RT
    reverse transcription
    PCR
    polymerase chain reaction
    bFGF
    basic fibroblast growth factor
    G-CSF
    granulocyte colony-stimulating factor
    PDGF
    platelet-derived growth factor
    ELISA
    enzyme-linked immunosorbent assay
    PI3K
    phosphatidylinositol 3-kinase
    IKK
    IκBα kinase
    GSK
    glycogen synthetase kinase
    SP600125
    anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone
    PD98059
    2′-amino-3′-methoxyflavone.

  • Received March 11, 2010.
  • Accepted May 17, 2010.

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Research ArticleCELLULAR AND MOLECULAR

Mammalian Target of Rapamycin Pathway Promotes Tumor-Induced Angiogenesis in Adenoid Cystic Carcinoma: Its Suppression by Isoliquiritigenin through Dual Activation of c-Jun NH2-Terminal Kinase and Inhibition of Extracellular Signal-Regulated Kinase

Zhi-Jun Sun, Gang Chen, Wei Zhang, Xiang Hu, Cong-Fa Huang, Yu-Fan Wang, Jun Jia and Yi-Fang Zhao
Journal of Pharmacology and Experimental Therapeutics August 1, 2010, 334 (2) 500-512; DOI: https://doi.org/10.1124/jpet.110.167692
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