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Mol Cancer Ther. 2006;5:129-137
© 2006 American Association for Cancer Research

Novel angiogenic inhibitor DN-9693 that inhibits post-transcriptional induction of connective tissue growth factor (CTGF/CCN2) by vascular endothelial growth factor in human endothelial cells

Seiji Kondo1,2, Noriko Tanaka3, Satoshi Kubota1, Yoshiki Mukudai1, Gen Yosimichi1, Toshio Sugahara2 and Masaharu Takigawa1

Departments of 1 Biochemistry and Molecular Dentistry and 2 Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan and 3 Research Planning and Administration Department, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan

Requests for reprints: Masahura Takigawa, Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8525, Japan. Phone: 81-86-235-6645; Fax: 81-86-235-6649. E-mail: takigawa{at}md.okayama-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Connective tissue growth factor (CTGF/CCN2) is a potent angiogenic factor. In this report, we describe for the first time that vascular endothelial growth factor (VEGF)–mediated induction of the ctgf/ccn2 gene was a post-transcriptional event that was inhibited by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. Steady-state mRNA levels of ctgf/ccn2 were remarkably increased by VEGF in a concentration-dependent manner, whereas the activity of the ctgf/ccn2 promoter was not responsive to VEGF as confirmed by a reporter gene assay and quantitative real-time PCR analysis. By employing a RNA degradation assay, we eventually found that the observed increase in the ctgf/ccn2 mRNA level was due to an increased stability of the mRNA induced by VEGF. DN-9693 at a dose of 0.1 to 2 ng/mL did not affect basal levels of ctgf/ccn2 mRNA; however, enhancement of ctgf/ccn2 mRNA expression by VEGF was specifically inhibited by DN-9693. Of importance, the inhibitory effects could be also ascribed to post-transcriptional regulation, because the VEGF-mediated increase in stability of ctgf/ccn2 mRNA was suppressed by DN-9693. Furthermore, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of mitogen-activated protein kinase pathways and found that DN-9693 blocked the activation of these pathways by VEGF. These results suggest that VEGF increases ctgf/ccn2 mRNA stability through mitogen-activated protein kinase–mediated intracellular signaling cascade(s), which can be inhibited posttranscriptionally by a novel angiogenic inhibitor, DN-9693, in human umbilical vein endothelial cells. [Mol Cancer Ther 2006;5(1):129–37]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Metastasis, the spread and growth of tumor cells to distant organs, is a major problem in cancer therapy. This process is dependent on neovascular formation from host tissues, suggesting that angiogenesis is a useful target of cancer treatment (1–3). To establish an angiostatic therapeutic strategy, it is critical to develop highly effective antiangiogenic agents. Indeed, synthetic or endogenous inhibitors of angiogenesis, such as TNP-470, SU-5416, angiostatin, and endostatin, have been reported to exhibit antiangiogenic and antimetastatic activities and nominated for clinical trials (4–6), and bevacizumab, a recombinant human monoclonal antibody specific for vascular endothelial growth factor (VEGF), was approved and launched for the first-line treatment of colon cancer (7, 8).

Thrombocytosis can be primary or secondary to a variety of illnesses (9). It is due to several disorders that cause a reactive stimulation of platelet production, familial mutations, or essential thrombocythemia and other myeloproliferative disorders. Thrombocytosis is also a frequent finding in cancer patients (9). The progression of cancer is associated with hypercoagulability, which results from the direct influences of tumor cells and other diverse indirect mechanisms. In this process, activated platelets serve as procoagulant surfaces amplifying the coagulation reactions. To prevent thrombotic complications, Anagrelide (Agrylin, Bristol-Myers Squibb, Princeton, NJ), a cyclic AMP phosphodiesterase inhibitor, is used to date. It had been firstly developed as a platelet aggregation inhibitor. However, it has been thereafter developed and launched for the treatment of thrombocythemia, because it unexpectedly caused the decrease in the number of platelets in clinical trials (10). One of the mechanisms for thrombocytopenic effect is known that Anagrelide suppresses megakaryocyte proliferation and differentiation. DN-9693, an analogue of Anagrelide, originated by Daiichi Pharmaceutical Co. Ltd. (Tokyo, Japan), was developed as a platelet aggregation inhibitor in the 1980s, but its development ceased. In the1990s, Tanaka and Aonuma unexpectedly found that DN-9693 as well as Anagrelide inhibited the proliferation of human endothelial cells induced by VEGF and/or basic fibroblast growth factor (bFGF). Since then, we have been pursuing the possibility of applying this drug to antineoplastic therapeutics due to its antiangiogenic effects.

We reported previously remarkably high levels of connective tissue growth factor (CTGF/CCN2) in human platelets released during the coagulation process (11). CTGF/CCN2 is a cysteine-rich secretory protein of 36 to 38 kDa, which is composed of 349 amino acid residues, and its gene belongs to the CCN family, which consists of cef10/cyr61, ctgf/fisp12, nov, and several recently reported genes, such as elm1/wisp1, ctgf3/ctgfL/wisp2/cop1, and wisp3 (12–17). CTGF/CCN2 modulates the effects of a variety of cytokines and has pleurotropic functions. It is of note that this factor not only mediates the adhesion of platelets and monocytes and is involved in wound healing and fibrosis but also promotes the growth and differentiation of vascular endothelial cells, osteoblasts, and chondrocytes in vitro as well as angiogenesis in vivo (18–24). Because platelets are a rich source of this endogenous angiogenic factor and play an important role in tumor-associated thrombocytosis, antiplatelet strategy may be effective against the development and metastasis of tumors through a dual mechanism. These findings together provide a rationale for the application of DN-9693 for antiangiogenic therapy in cancer patients. However, the precise mechanism of the antiangiogenic action of DN-9693 is mostly unknown. Therefore, in this study, we examined the effects of DN-9693 on VEGF-induced CTGF/CCN2 expression in human umbilical vein endothelial cells (HUVEC).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DN-9693 was synthesized and supplied by Daiichi Pharmaceutical Co. Ltd. The chemical structure of DN-9693 is shown in Fig. 1 . For in vitro experiments, DN-9693 was dissolved in DMSO at a concentration of 70 µg/mL for the stock solutions and kept at –80°C before use. Actinomycin D and recombinant human bFGF were purchased commercially from Sigma (St. Louis, MO). Recombinant human VEGF165 was obtained from Genzyme/Techne (Minneapolis, MN). Anti-phospho-p42/p44 mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase (ERK) 1/2] and anti-phospho-p38 were obtained from Promega (Madison, WI), and anti-p42/p44 MAPK (ERK1/2), anti-phospho–c-Jun NH2-terminal kinase, and anti–c-Jun NH2-terminal kinase were from Cell Signaling Technology (Beverly, MA). Anti-p38 was from Calbiochem (Bad Soden, Germany).


Figure 1
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  		Figure 1. Chemical structure of DN-9693. Formula: C15H18N4O; molecular weight: 361.27.

 
Cell Culture
HUVECs derived from human umbilical cords were purchased from Morinaga Institute of Biological Science (Kanagawa, Japan) or BioWhittaker (Walkersville, MD) and used between passages 3 and 7. These cells were cultured at 37°C in a 5% CO2 atmosphere in HCM medium (Nissui Pharmacuetical Co. Ltd., Tokyo, Japan) or EBM-2 complete medium (Clonetics, San Diego, CA). The human microvascular vein endothelial cells were purchased from Morinaga Institute of Biological Science and used between passages 3 and 7. P388 cells derived from a mouse leukemia and PC-6 cells derived from a human lung cancer were obtained from Daiichi Pharmaceutical Co. Ltd. They were cultured at 37°C with 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS).

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay
HUVECs (1 x 103/well) and human microvascular vein endothelial cells (1 x 103/well) were seeded with HCM medium with 0.2% FBS in 96-well plates (Falcon Laboratories, McLean, VA). After 6 hours, the cells were treated with VEGF (2.5 ng/mL) or bFGF (0.3 ng/mL) and with various concentrations of DN-9693 and then further cultured for 4 days. P388 cells (1 x 103/well) and PC-6 cells (1 x 103/well) were seeded with serum-deprived DMEM in 96-well plates. After 6 hours, the cells were treated with FBS (final concentration, 10%) and various concentrations of DN-9693 and further cultured for 2 days. The proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) assay as described previously (19). Briefly, 0.5% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (10 µL) in distilled water was added to 100 µL medium in each well. After incubation for 4 hours at 37°C, the medium was removed, and the cells were lysed in 0.04 mol/L HCl 2-propanol. Then, the absorbance of the lysate was measured at a wavelength of 570 nm (excitation, 630 nm).

Tube Formation Assay
Matrigel (50 µL; Collaborative Biomedical Products, Bedford, MA) was pipetted into each well of 96-well plates and polymerized for 30 minutes at 37°C. After trypsin treatment, HUVECs were harvested onto the layer of Matrigel at a density of 6 x 103 per well with M199 medium supplemented with 20% FBS and various concentrations of DN-9693. After 18 hours, the cultures were photographed (x40). The area covered by the tube network was determined using an optical imaging technique and quantified using Quantimet (Leica Cambridge Ltd., Cambridge, England).

Plasmid Constructs and DNA Transfection
A ctgf/ccn2 promoter-driven firefly luciferase expression plasmid, termed pTS589, was obtained from Japan Tobacco, Inc. (Yokohama, Japan; ref. 23). Subconfluent HUVECs in six-well plates were transfected with the plasmid described above (1.0 µg), and an internal control, pRL-TK (0.5 µg), contains a Renilla luciferase gene under the control of the herpes simplex virus thymidine kinase gene promoter (Promega) using a cationic liposome reagent, Fugene 6 transfection reagent (Roche, Basel, Switzerland). Twenty-four hours after transfection, the cells were replenished with EBM-2 containing just 0.2% FBS and free of other supplements and cultured for further 8 hours. The cells were then stimulated with VEGF or DN-9693. After 6 hours, the cells were lysed in 500 µL of a passive lysis buffer (Promega), and the cell lysate was directly used in the luciferase assay system as described below.

Luciferase Assay
The dual luciferase system (Promega) was used for the sequential measurement of firefly and Renilla luciferase activities with the specific substrates beetle luciferin and coelenterazine, respectively. Quantification of both luciferase activities and calculation of relative ratios were carried out manually with a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA).

RNA Extraction and Quantitative Real-time PCR
Subconfluent HUVECs in 6-cm dishes were replenished with EBM-2 containing 0.2% FBS and free of other supplements and cultured a further 8 hours. Cells were treated with various concentrations of VEGF or DN-9693 and cultured another 6 hours (Figs. 2A and 3A ). Cells were pretreated for 1 hour with 2 ng/mL DN-9693 before being exposed to VEGF (25 ng/mL). After 6 hours, the cells were harvested (Fig. 4A ). Total cellular RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's directions. Total cellular RNA was isolated as described above, and a cDNA mixture was generated by reverse transcription using oligo(dT) and an avian myeloblastosis virus reverse transcriptase (Invitrogen, San Diego, CA). The cDNA was used as a template in the subsequent real-time PCR analysis using FastStart DNA Master SYBR Green I and a LightCycler apparatus (Roche Diagnostics, Mannheim, Germany). Primers used were 5'-TGACTGCCCCTTCCCGAGAA-3' and 5'-TCTTCCAGTCGGTAGGCAGCTAGG-3' for ctgf/ccn2 and 5'-GATCATTGCTCCTCCTGAGC-3' and 5'-ACTCCTGCTTGCTGATCCAC-3' for ß-actin. An initial denaturation at 95°C for 10 minutes was followed by 45 cycles of denaturation at 95°C for 15 seconds, annealing at 55°C for 10 seconds, and elongation at 72°C for 10 seconds. The fluorescence intensity of the double strand–specific SYBR Green I, reflecting the amount of PCR product, was monitored at the end of the elongation step. The ß-actin mRNA levels were used to normalize the sample cDNA content.


Figure 2
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  		Figure 2. Effects of VEGF on ctgf/ccn2 mRNA expression. A, dose-dependent induction of ctgf/ccn2 mRNA expression 6 h after stimulation with VEGF. Real-time RT-PCR quantification after normalization to the control (ß-actin) signals in two sets of experiments. Columns, average; bars, SD. *, P < 0.05. B, effects of VEGF on ctgf/ccn2 promoter activity. HUVECs were cotransfected with 1 µg of the ctgf/ccn2 promoter-firefly luciferase reporter plasmid (pTS589) and 0.5 µg thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK, internal control; Promega), and the cells were allowed to recover for 24 h after transfection. After their recovery, the transfected cells were stimulated with VEGF and incubated a further 6 h, and the cells were then assayed for luciferase activities. F/R, relative value of the measured luminescence of firefly luciferase versus Renilla luciferase. Columns, mean of three experiments; bars, SD.

 

Figure 3
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  		Figure 3. Effects of DN-9693 on the basal levels of ctgf/ccn2 mRNA. A, ctgf/ccn2 mRNA levels showed no change after treatment with DN-9693 at up to 2 ng/mL in HUVECs. Real-time RT-PCR quantification after normalization to the control (ß-actin) signals in two sets of experiments. Columns, average; bars, SD. B, no significant effects of DN-9693 on the ctgf/ccn2 promoter activity in HUVECs was found regardless of the dose. The ctgf/ccn2 promoter-luciferase reporter plasmid (pTS589) or pGL3-control (cont; Promega) and pRL-TK were used to cotransfect HUVECs, which were then incubated in the presence of various concentrations of DN-9693. Columns, mean of three experiments; bars, SD. C, effect of DN-9693 on rate of ctgf/ccn2 transcription in HUVECs. Nuclei from the FBS (0.04%, positive control)–, VEGF-, and/or DN-9693-treated cells were isolated and used for run-on transcription assays. Autoradiograms of labeled transcripts representing ctgf/ccn2 and g3pdh as an internal control and pGEM3Zf(+) as a negative control. Top, representative of two individual experiments; bottom, relative value of ctgf/ccn2 transcripts normalized to the g3pdh level. Columns, average; bars, SD. At the same time, RNA from VEGF- or DN-9693-treated cells was isolated and used for real-time RT-PCR. Results were consistent with Fig. 2A and with A. (VEGF increased ctgf/ccn2 mRNA levels by ~2-fold compared with that in control cells, and DN-9693 caused no change in ctgf/ccn2 mRNA levels.) D, DN-9693 failed to inhibit serum-induced ctgf/ccn2 expression in HUVECs. Autoradiograms of labeled transcripts representing ctgf/ccn2 and g3pdh as an internal control and pGEM3Zf(+) as a negative control. Induction of ctgf/ccn2 transcription by FBS (positive control) was done at the dose of 0.04%.

 

Figure 4
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  		Figure 4. DN-9693 inhibited VEGF-induced ctgf/ccn2 mRNA expression and protein production by HUVECs. A, DN-9693 remarkably inhibited VEGF-induced ctgf/ccn2 gene expression in a dose-dependent manner, with a complete blockade of the effect of 25 ng/mL VEGF at 2 ng/mL. HUVECs were pretreated for 1 h with various concentrations (0.1, 2, or 10 ng/mL) of DN-9693 before being exposed to VEGF (25 ng/mL). After 6 h, the cells were harvested. Real-time RT-PCR quantification after normalization to the control (ß-actin) signals in two experiments. *, P < 0.05. B, CTGF/CCN2 production increased by VEGF was also inhibited by DN-9693. Quantification of the growth factor was done by using an ELISA system. *, P < 0.05. Columns, mean of duplicates; bars, SD.

 
Nuclear Run-on Analysis
Nuclear run-on assays were carried out according to an established protocol (25). Nuclei isolated from the FBS (0.04%, positive control)–, VEGF (25 ng/mL)–, and DN-9693 (0.5 or 2 ng/mL)–stimulated cells were suspended in 50 mmol/L Tris-HCl (pH 8.0), 40% glycerol, 5 mmol/L MgCl2, and 0.1 mmol/L EDTA, and aliquots were incubated at 30°C for 2 hours with a mixture of ATP, CTP, and GTP (2.5 mmol/L each) and 100 µCi [{alpha}-32P]UTP (3,000 Ci/mmol; Amersham Pharmacia, Aylesbury, United Kingdom) in a reaction buffer consisting of 10 mmol/L Tris-HCl (pH 8.0), 5 mmol/L MgCl2, and 300 mmol/L KCl. After digestion with RQ1 DNase and proteinase K (Invitrogen, Carlsbad, CA), the radiolabeled transcripts were extracted using Isogen according to the manufacturer's directions and further purified on a spin column. The pGEM3Zf(+) human ctgf/ccn2 vector containing a full-length ctgf/ccn2 open reading frame was used as a probe for the ctgf/ccn2 gene. The probe for the human g3pdh gene was obtained by conducting a reverse transcription-PCR (RT-PCR) using total cellular RNA of HCS-2/8 (human chondrosarcoma) as a template. The nucleotide sequences of the sense and antisense primers for human g3pdh were 5'-AAAGAAGATGCGGCTGACTG-3' and 5'-TCCAGGGGTCTTACTCCTTG-3', respectively. The amplicons were subcloned into pGEM T-Easy (Promega) using a TA cloning method. For the preparation of membranes, the plasmids containing human g3pdh and ctgf/ccn2 were linearized by PstI and EcoRI, respectively, whereas pGEM3Zf(+), as a background control, was linearized by HindIII. The plasmids were subjected to alkaline denaturation, and 10 µg of each were dot blotted onto a nylon membrane using a Bio-Dot apparatus (Bio-Rad, Hercules, CA). Radiolabeled nuclear transcripts (107 counts/min/mL) were hybridized to the blotted membranes at 42°C for 72 hours in a hybridization buffer consisting of 5x SSC (pH 7.0), 5x Denhardt's solution (1x Denhardt's solution = 0.002% polyvinylpyrolidone, 0.02% Ficoll 400, and 0.02% bovine serum albumin), 2% SDS, 50% formamide, and 100 mg/mL sonicated salmon testis DNA (Sigma). After hybridization, the membranes were washed at 65°C for 20 minutes twice in a wash buffer composed of 0.1x SSC (pH 7.0) and 0.1% SDS and then subjected to autoradiography.

Analysis of ctgf/ccn2 mRNA Half-life
Subconfluent HUVECs in 6-cm dishes were replenished with EBM-2 containing 0.2% FBS and free of other supplements and cultured for 8 hours. The cells were exposed to PBS or VEGF (25 ng/mL) for 4 hours before measurements of mRNA stability. Transcription was inhibited by the addition of actinomycin D (5 µg/mL) and harvested for the indicated time (Fig. 5A ). For Fig. 5B, cells were pretreated for 1 hour with 2 ng/mL DN-9693 or DMSO (control) before exposure to PBS or VEGF (25 ng/mL).


Figure 5
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  		Figure 5. DN-9693 inhibited the stabilizing effect of VEGF on ctgf/ccn2 mRNA. A, degradation of ctgf/ccn2 mRNA in HUVECs in the presence or absence of 25 ng/mL VEGF. The relative amount of ctgf/ccn2 mRNA remaining after treatment with actinomycin D (Act D; 5 µg/mL) for the indicated times was quantified by real-time RT-PCR analysis and displayed as a percentage versus the control sample at time 0. B, degradation of VEGF-treated ctgf/ccn2 mRNA in HUVECs in the presence or absence of 2 ng/mL DN-9693. DN-9693 at a dose of 2 ng/mL inhibited the VEGF-enhanced stability of ctgf/ccn2 mRNA to nearly the basal level.

 
ELISA
Subconfluent HUVECs in 10-cm dishes were replenished with EBM-2 containing 0.2% FBS and free of other supplements and cultured another 8 hours. Cells were pretreated for 1 hour with various concentrations (0.1 or 2 ng/mL) of DN-9693 before being exposed to VEGF (25 ng/mL). After 12 hours, the cells were harvested, and ELISA was done. A sandwich ELISA for CTGF/CCN2 was carried out as described previously (26–28). This system was developed with two different anti-human CTGF/CCN2 antibodies, one specific for the CT domain and the other specific for the VWC domain. After the reaction with these antibodies, the amount of CTGF/CCN2 in 50 µL of each sample was represented by the intensity of the fluorescence emitted by the enzymatic reaction in each well of the ELISA plate at the wavelength 460 nm (excitation, 360 nm), which was determined by using a Cytoflor 2300 system (Millipore Corp., Billerica, MA) after a 10-minute incubation at room temperature.

Western Blot Analysis
Subconfluent HUVECs in 3.5-cm dishes were replenished with EBM-2 containing 0.2% FBS and free of other supplements and cultured for 8 hours. The cells were exposed to VEGF (25 ng/mL) and then were harvested after indicated time. For inhibitory assays of the kinase pathways, cells were pretreated for 1 hour with 2 ng/mL DN-9693 before being exposed to VEGF. Total cellular protein was prepared by lysing cells in a lysis buffer [20 mmol/L Tris-HCl (pH 8.0) containing 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 1 mmol/L Na3VO4, 5% glycerol, 40 mmol/L ammonium molybdate, and 1 mmol/L phenylmethylsulfonyl fluoride]. The proteins in the samples were separated by 4% to 20% gradient SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were blocked for 30 minutes at room temperature with 5% nonfat dry milk in a TBS (pH7.5) containing 0.1% Tween 20 and then incubated with each specific antibody for 24 hours at 4°C. The membranes were then incubated with a secondary antibody (horseradish peroxidase–conjugated anti-rabbit IgG, DAKO, Trappes, France), and the signal was visualized by use of an enhanced chemiluminescence system (Amersham Biosciences, Uppsala, Sweden).

Statistical Analysis
Unless otherwise specified, all experiments were repeated at least twice, with similar results. Statistical analysis was done with Dunnett's test (Fig. 5B) or Student's t test (Figs. 2A and 4) if necessary. Data were expressed as mean ± SD, and differences were considered significant at P < 0.05 and P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DN-9693 Inhibits Growth Factor–Induced Proliferation and Tube Formation of Endothelial Cells
To determine the antiangiogenic activity of DN-9693 in vitro, its inhibitory effect on the VEGF- or bFGF-induced proliferation of endothelial cells was evaluated. Six hours after seeding, HUVECs or human microvascular vein endothelial cells were treated with various concentrations of DN-9693 and VEGF or bFGF (2.5 and 0.3 ng/mL, respectively) and cultured for 4 days. As shown in Table 1 , DN-9693 inhibited the VEGF- or bFGF-induced endothelial cell proliferation in a dose-dependent manner, with half-maximal inhibition at 1.6 and 2.2 ng/mL, respectively. In addition, we examined whether DN-9693 interfered directly with tumor cell proliferation. Interestingly, DN-9693 even at high concentrations (>50,000 ng/mL) had no inhibitory effect on P388 or PC-6 cell proliferation. These results suggested that DN-9693 specifically inhibited endothelial cell growth. Subsequently, the effect of DN-9693 on morphologic differentiation of endothelial cells was investigated using Matrigel. DN-9693 effectively abrogated the width and length of endothelial tubes in a concentration-dependent manner (data not shown). Half-maximal inhibition was seen at a concentration of 330.3 ng/mL. These results show that DN-9693 has an ability to block growth factor–induced in vitro angiogenesis.


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  		Table 1. Specific inhibitory effects of DN-9693 on the growth of endothelial cells

 
VEGF Increases ctgf/ccn2 mRNA Expression in HUVECs
It is well known that endothelial cell proliferation is involved in extracellular matrix remodeling. Because VEGF induces a growth factor, CTGF/CCN2, which stimulates production of extracellular matrix and fibrosis in retinal vascular cells (29), we examined the effects of VEGF on the expression of ctgf/ccn2 in HUVECs. The response of ctgf/ccn2 mRNA expression to VEGF was studied after 6 hours of stimulation by quantitative real-time PCR. As shown in Fig. 2A, VEGF increased ctgf/ccn2 mRNA levels in a dose-dependent manner, with significant increases observed at 25 ng/mL in HUVECs (~2.5-fold; P < 0.05).

CTGF/CCN2 Gene Induction by VEGF Is Not Due to the Activation of Transcription but Occurs at the Post-transcriptional Level
Next, we evaluated the activity of the ctgf/ccn2 promoter on addition of VEGF by using a previously described reporter plasmid containing 800 bp of the ctgf/ccn2 proximal promoter (23, 27). As shown in Fig. 2B, the reporter gene expression showed no change regardless of the doses of VEGF. This result showed that activation of the prototypic ctgf/ccn2 promoter did not occur together with the induction of ctgf/ccn2 gene expression by VEGF. Because the role of transcription cannot be ruled out by only using transient transfection with a promoter, a nuclear transcript run-on assay was done. As shown in Fig. 3C, transcription rates were the same in nuclei from either untreated control or VEGF-stimulated cells in contrast to the 2- to 3-fold increase in the steady-state ctgf/ccn2 mRNA level (Fig. 2A). Therefore, the effect of VEGF to increase the ctgf/ccn2 mRNA level is predominantly post-transcriptional in HUVECs.

Effects of DN-9693 on ctgf/ccn2 mRNA Levels and Promoter Activity
Because CTGF/CCN2 is a potent angiogenic factor (18–22), we examined the effects of DN-9693 on the expression of ctgf/ccn2 in HUVECs. As shown in Fig. 3A, ctgf/ccn2 mRNA levels showed no change after treatment with DN-9693 at up to 2 ng/mL in comparison with those in HUVECs. To confirm that altered ctgf/ccn2 transcription did not occur in the presence of DN-9693, we did a transient reporter gene assay using the plasmid described in Fig. 2. As shown in Fig. 3B, the promoter activity showed no change in the presence or absence of DN-9693 in HUVECs. Furthermore, a nuclear transcript run-on assay was done to confirm the effects of DN-9693 on ctgf/ccn2 mRNA levels. As shown in Fig. 3C, transcription rates were the same in nuclei from either untreated control or DN-9693-stimulated cells. Therefore, no effect of DN-9693 itself on ctgf/ccn2 mRNA levels was observed in HUVECs. Additionally, we examined whether any inhibitory effect of DN-9693 can be seen on induced ctgf/ccn2 transcription. Transcription of ctgf/ccn2 was induced by FBS at a dose of 0.04%, and the effect of DN-9693 was evaluated. As shown in Fig. 3D, transcription rates were the same in nuclei from FBS-stimulated cells either with or without DN-9693. Therefore, DN-9693 does not even inhibit induced ctgf/ccn2 expression at transcriptional level(s) in HUVECs.

DN-9693 Inhibits VEGF-Induced ctgf/ccn2 Expression in HUVECs
To evaluate the influence of DN-9693 on the effects of VEGF on ctgf/ccn2 gene expression, we pretreated HUVECs with specific concentrations of DN-9693 before stimulation with 25 ng/mL VEGF and then cultured them for 6 hours. As a result, DN-9693 significantly inhibited VEGF-induced ctgf/ccn2 expression in a dose-dependent manner. Indeed, 2 ng/mL DN-9693 completely blocked the effect of 25 ng/mL VEGF (Fig. 4A). Therefore, this concentration was determined to be sufficient to antagonize the effect of VEGF and chosen for subsequent mRNA degradation experiments.

DN-9693 Inhibits VEGF-Induced CTGF/CCN2 Protein Production in HUVECs
To determine if the effects of DN-9693 on VEGF-induced ctgf/ccn2 mRNA expression correlated with the protein level, CTGF/CCN2 protein production was assessed by ELISA. As shown in Fig. 4B, VEGF significantly increased levels of CTGF/CCN2 protein produced by HUVECs, although the increase did not reach the level observed for ctgf/ccn2 mRNA. DN-9693 inhibited the VEGF-induced CTGF/CCN2 protein production in a dose-dependent manner.

VEGF Prolongs the Half-life of ctgf/ccn2 mRNA in HUVECs, an Effect Inhibited by DN-9693
To determine whether the up-regulation of ctgf/ccn2 mRNA expression by VEGF indeed was due to increased mRNA stability, we first treated HUVECs with VEGF (25 ng/mL) for 4 hours and then did actinomycin D (5 µg/mL) chase experiments for up to 2 hours. As shown in Fig. 5A, in the absence of VEGF, the basal half-life of ctgfccn2 mRNA was relatively short (1 hour). However, in the presence of VEGF, the half-life was increased over 2 hours. Together with the findings displayed in Figs. 2A and B and 3C, the ability of VEGF to increase ctgf/ccn2 mRNA levels can be ascribed predominantly to post-transcriptional regulation in HUVECs. Next, we examined the influence of DN-9693 on the stabilizing effect of VEGF on ctgf/ccn2 mRNA in HUVECs. As expected, the effect of VEGF on the half-life of ctgf/ccn2 mRNA was reversed by DN-9693 at a dose of 2 ng/mL. These findings suggest that DN-9693 specifically inhibits the stabilization of ctgf/ccn2 mRNA by VEGF.

DN-9693 Inhibits VEGF-Induced Activation of Three Subgroups of MAPK Pathways
To understand the molecular mechanism by which DN-9693 inhibits VEGF-induced angiogenesis, we investigated the effects of DN-9693 on VEGF-induced activation of three subgroups of MAPK pathways. Firstly, we examined which MAPK pathways were activated by VEGF. As shown in Fig. 6A , the VEGF activation of three subgroups of MAPK pathways (p38 kinase, ERK, and c-Jun NH2-terminal kinase) was observed in HUVECs. Indeed, phosphorylation of any MAPKs became detectable as early as 5 to 15 minutes after the stimulation by VEGF. Next, we employed inhibition assays of these kinases to determine which MAPK pathways were blocked by DN-9693. As the effects of VEGF on the phosphorylation of the MAPKs were prominently observed within 15 minutes (Fig. 6A), the inhibition assays by DN-9693 were also done within such a period. As shown in Fig. 6B, DN-9693 significantly diminished VEGF activation of all of the three subgroups of MAPK pathways in a dose-dependent manner, with enough effects at 2 ng/mL. These results indicate that DN-9693 acts as a blocker of these kinase pathways activated by VEGF in HUVECs.


Figure 6
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  		Figure 6. DN-9693 inhibits VEGF-induced activation of three subgroups of MAPK pathways. A, signal transduction profile in VEGF-treated HUVECs. HUVECs were cultured after the stimulation by VEGF (25 ng/mL) for the indicated times and then phosphorylated or total ERK1/2, phosphorylated or total p38, and phosphorylated or total c-Jun NH2-terminal kinase 1/2 (JNK1/JNK2) were analyzed by Western blot analysis using their respective specific antibodies. B, effects of DN-9693 pretreatment. Pretreatment of HUVECs with DN-9693 before the VEGF treatment blocked the VEGF activation of almost all of the three subgroups of MAPK pathways at a dose of 2 ng/mL.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Antiangiogenesis has become one of the most promising approaches in the development of anticancer drugs (1–3). It seems that most inhibitors of angiogenesis specifically target the newly formed vasculature in tumors, not quiescent blood vessels, indicating little possibility of side effects (4–6). In addition, because these endothelial cell compartments are normal and genome stable, it is not likely they will develop drug resistance problems. This is an important step forward in cancer therapy as other current anticancer therapies, including chemotherapy and radiotherapy, generally have severe adverse events. Therefore, there is no doubt that blockage of the tumor blood supply will be beneficial for cancer patients. Indeed, many currently available angiogenic inhibitors have produced remarkable antitumor effects in animal models (5, 6). However, although these inhibitors have been found to be potent anticancer agents, the underlying mechanisms of their actions are relatively poorly understood. Without knowing exactly how they function at the molecular level, it is risky to apply them clinically and difficult to predict the clinical consequence in human trials.

The present study provides novel evidence supporting the ability of DN-9693 to act as an active antiangionenic agent that causes suppression of the proliferation of human endothelial cells in vitro. One of the molecular mechanisms clarified was that VEGF-mediated induction of the ctgf/ccn2 gene was inhibited post-transcriptionally through the control of ctgf/ccn2 mRNA stability in HUVECs. Furthermore, DN-9693 inhibited three subgroups of MAPKs activated by VEGF in HUVECs. Recently, involvement of RhoA GTPase and p38 MAPK in the mechanism of post-transcriptional regulation of ctgf/ccn2 mRNA stability was suggested (30). In addition, in mesangial cells, ras/MEK/ERK, tyrosine kinase, and protein kinase C activities were reported to be necessary for the induction of CTGF/CCN2 by transforming growth factor-ß (31). The role of MAPK pathways in induction of ctgf/ccn2 gene expression by cytokine seems to be different depending on cell types and types of stimuli to which the cells are exposed. Although the role of MAPK pathways in the induction of ctgf/ccn2 by VEGF was not directly investigated in this study, it is plausible that DN-9693 may inhibit VEGF-induced ctgf/ccn2 expression through these MAPK pathways. Of note, we showed that a cis-element in the 3'-untranslated region of chicken ctgf/ccn2 mRNA and trans-factor counterpart(s) play an important role in the post-transcriptional regulation by determining the stability of ctgf/ccn2 mRNA (25). The MAPK pathways may contribute to VEGF-induced ctgf/ccn2 expression by transmitting phosphorylation signal(s) to stabilize the mRNA, which may be mediated by 3'-untranslated region AU-rich motif. Therefore, it is of our current interest to inhibit these signal pathways by DN-9693 and to examine if it affects the binding profile of trans-acting protein(s) that recognize the 3'-untranslated region of ctgf/ccn2 mRNA. Clearer understanding of the mechanism of this regulation requires the identification and characterization of the trans-acting factor(s) that interact with an element in the 3'-untranslated region. Work is in progress to identify these proteins.

To be effective against cancer, an antiangiogenic agent would have to be administered continuously for a long time, suggesting that oral administration is the first choice. One should note that long-term multiple administration of a platelet-lowering drug, Anagreride, is well tolerable. Also in this study, an analogue of Anagreride, DN-9693, produced remarkable antiangiogenic effects with no significant side effects at relatively low doses, up to 2 ng/mL. Because combinations of various angiogenic inhibitors should be used for the treatment of human cancers, the effectiveness of DN-9693 at low doses may be a critical advantage when used with other antiangiogenic agents.

From a biological perspective, the effects of VEGF on ctgf/ccn2 mRNA are potentially important not only for endothelial proliferation but also for tumor progression. An early tumor may secrete a single angiogenic factor, whereas a large progressive tumor can produce many angiogenic factors (32). Indeed, >50% of newly diagnosed breast cancers produce only VEGF as an angiogenic stimulator. However, along with subsequent tumor progression, recurrence, and metastasis, the production of other angiogenic factors, including FGF-2, transforming growth factor-ß, and platelet-derived growth factor, is initiated (32). Therefore, the application of a single angiogenic factor inhibitor may well encounter complications due to the development of drug resistance. VEGF might regulate the expression of CTGF/CCN2 upstream of the cascade, and both of them could play a central role in tumor progression and metastasis. It is also possible that other angiogenic factors are awaiting the command downstream of the cascade.

We confirmed that DN-9693 inhibits VEGF-induced CTGF/CCN2 expression in endothelial cells. Because DN-9693 shuts off this cascade in endothelial cells, it may be useful as an angiogenic inhibitor of least possibility of side effects in cancer therapy. Because the underlying mechanisms of angiogenic inhibitors are complex and require many signaling players to coordinately suppress angiogenesis, it is essential to elucidate how endothelial cell apoptosis, the antagonistic effect of the growth factor–induced signaling pathway, the regulation of oncogenes and tumor suppression genes, and the repression of endothelial cell cycles are involved in the antiangiogenic activities of these inhibitors. Further research is required to clarify the mechanisms of the antiangiogenic action of DN-9693.


   Acknowledgments
 
We thank Dr. Takanori Eguchi for his helpful suggestions, Kazumi Ohyama for technical assistance, and Yuki Nonami for secretarial assistance.


   Footnotes
 
Grant support: Grants-in-Aid for Scientific Research (S)(M. Takigawa) and (C)(S. Kubota), and Exploratory Research (M. Takigawa) of the Ministry of Education, Science, Sports, and Culture of Japan, Naito Foundation (M. Takigawa), Nakatomi Health Science Foundation (S. Kubota and M. Takigawa), Foundation for Growth Science in Japan (M. Takigawa), and Sumitomo Foundation (M. Takigawa).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: S. Kondo is a research fellow of the Japan Society for the Promotion of Science.

Received 4/ 4/05; revised 10/ 3/05; accepted 10/28/05.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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