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CARDIOVASCULAR
Pharmacological Institute (S.-L.P., C.-Y.P., S.-W.W., Y.-L.C., C.-M.T.) and School of Pharmacy (J.-H.G.), College of Medicine, National Taiwan University, Taipei, Taiwan; MDS Pharma Service, Taipei, Taiwan (F.-C.C.); Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan (S.-C.K.); and Yung-Shin Pharmaceutical Industry Co., Ltd., Taichung, Taiwan (J.-H.C., F.-Y.L.)
Received February 17, 2005; accepted March 18, 2005.
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Abstract |
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translocation using Western blot analysis. The effect of YC-1 on angiogenesis in vivo was evaluated using the mouse Matrigel implant model. YC-1 administered orally in doses of 1 to 100 mg/kg/day inhibited VEGF- and bFGF-induced neovascularization in a dose-dependent manner over 7 days. These results indicate that YC-1 has antiangiogenic activity at very low doses. Moreover, in transplantable murine tumor models, YC-1 administered orally displayed a high degree of antitumor activity (treatment-to-control life span ratio > 175%) without cytotoxicity. YC-1 may be useful for treating angiogenesis-dependent human diseases such as cancer.
). Physiologically, angiogenesis plays a crucial role in embryonic development, placental implantation, and wound healing. In contrast, the same process also supports pathological conditions such as solid tumor growth, diabetic retinopathy, psoriasis, and rheumatoid arthritis. Complex and diverse cellular actions such as extracellular matrix degradation, proliferation and migration of endothelial cells, and morphological differentiation of endothelial cells to form tubes have all been implicated in angiogenesis (Bussolino et al., 1997). Although all of these processes are regulated under normal conditions, abnormal vascularization is clearly implicated in malignant tumor growth and metastasis. The extreme growth of tumors to volumes larger than a few cubic millimeters requires continuous recruitment of new blood vessels (Folkman, 1990). These newly synthesized blood vessels also provide a route for cancer cells to enter the systemic circulation and spread to distant organs (Fidler and Ellis, 1994). A growing number of angiogenic factors have been recognized as contributors to regulation of tumor angiogenesis. When a balance between angiogenic and angiostatic factors is disrupted, uncontrolled angiogenic factors such as growth factors, cytokines, lipid metabolites, and cryptic fragments of hemostatic proteins can be released from tumor cells. Among these angiogenesis-related factors are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) (Klein et al., 1997; Ferrara, 2001). These factors are produced by many pathologic (tumor) and normal cells, and they stimulate endothelial cell proliferation, sprouting, migration, and morphogenesis. They act via receptor tyrosine kinases and influence normal and abnormal angiogenesis (Friesel and Maciag, 1999; Cross and Claesson-Welsh, 2001).
YC-1 is a novel NO-independent type of soluble guanylyl cyclase (sGC) activator that mimics many of the known functions of NO and NO donors. YC-1 was discovered in Teng's laboratory (Ko et al., 1994). YC-1 and NO synergistically activate sGC in vitro, and YC-1 potentiates the effects of exogenous and endogenous carbon monoxide on sGC (Friebe et al., 1996). YC-1 at a high concentration level (e.g., >30 µM) induces these cellular and physiological functions via cGMP-dependent or independent pathways (Wang et al., 2002; Chien et al., 2003; Pan et al., 2004). However, we have not found published studies reporting an inhibitory effect of YC-1 on endothelial cells at a very low concentration. In this study, we determined that low levels of YC-1 suppressed endothelial cell proliferation, migration, and tube formation in vitro and inhibited VEGF- and bFGF-induced angiogenic signaling pathways. YC-1 also markedly inhibited neovascularization induced by VEGF and bFGF in vivo in the Matrigel plug implantation model. Finally, YC-1 prolonged the life-span of tumor-treated animals in an antitumor activity experiment without producing any apparent cytotoxicity. Taken together, these results suggest that YC-1 may inhibit tumor activity via the antiangiogenic properties demonstrated at a low level of exposure.
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Materials and Methods |
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), and cultured in 75-cm2 plastic flasks in M199 medium containing 20% fetal bovine serum (FBS) and 15 µg/ml endothelial cell growth supplements. Confirmation of cell identity as endothelial cells was provided by detection of CD31 (PECAM-1) by immunostaining. The human lung cancer cell line A549 was cultured in Dulbecco's modified Eagle's medium containing 10% inactivated FBS. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Medium was changed every 2 days, and cells were passaged after treatment with a solution of 0.05% trypsin/0.02% EDTA. Experiments were conducted on HUVECs that had gone through two to five passages. [3H]Thymidine Incorporation Assay. Confluent HUVECs were trypsinized, suspended in M199 medium supplemented with 20% FBS, and seeded at 1.0 x 104 cells per well into 96-well plates. After 24 h, the cells were washed twice with phosphate-buffered saline (PBS) and starved with 2% FBS-M199 medium for 24 h. The cells were then incubated with or without indicated reagents and growth factors (e.g., VEGF and bFGF at 10 ng/ml) for 24 h and harvested. Before the harvest, cells were incubated with [3H]thymidine (2 µCi/ml) for 4 h. Cells were harvested with Filter-Mate (PerkinElmer Life and Analytical Sciences, Boston, MA), and incorporated radioactivity was determined.
Migration Assay. Chemotactic migration of HUVECs was measured with a transwell migration apparatus as described previously by Pan et al. (2003). Cell migration was assayed in 24-well, 6.5-mm-internal diameter chamber cluster plates (8-µm pore size; Costar, Cambridge, MA). Briefly, HUVECs (1 x 105 cells/well) were suspended in 200 µl of serum-free M199 medium with 0.1% bovine serum albumin and loaded into the upper chamber of a transwell cluster plate coated with 0.1 mg/ml gelatin. VEGF or bFGF was diluted to 10 ng/ml in 0.6 ml of M199/0.1% bovine serum albumin and added to the lower wells of the chamber. YC-1 (3–30 µM) was added at the indicated concentration 1 h before the assay. The chambers were incubated for 24 h at 37°C in an atmosphere of 95% air and 5% CO2. At the end of incubation, cells were fixed and stained with hematoxylin. Nonmigrated cells on top of the filters were wiped off, filters were mounted, and migrated cells attached to the bottom of the filter were counted in six randomly chosen (x400) high-power fields. Cell migration was calculated as the difference between the number of migrated cells in the YC-1-treated samples and the number of migrated cells in the control samples.
Tube Formation Assay. HUVECs (2 x 105 cells) were seeded on a layer of polymerized Matrigel with or without YC-1 (3–30 µM) and VEGF or bFGF (10 ng/ml) in a chamber slide (Nalge Nunc International, Naperville, IL). Matrigel cultures were incubated at 37°C. After 24 h, cell morphology was evaluated using a phase-contrast microscope, and cells were photographed.
PKC Fractionation in HUVECs. Cells were homogenized in 0.2 ml of buffer A containing 20 mM HEPES (pH 7.5), 0.33 M sucrose, 2 mM EGTA, 2 mM EDTA, 5 mM dithiothreitol, 20 mM sodium fluoride, 0.1 mM sodium orthovanadate, 20 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.2 mM phenylmethyl sulfonyl fluoride. The cytosolic fraction (supernatant) was separated by centrifugation at 100,000g for 60 min. The pellet was resuspended in homogenization buffer A without sucrose but with 0.1% Triton X-100, followed by centrifugation and collection of the particulate fraction (supernatant). The samples were processed for SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis.
Western Blotting. After exposure of cells to indicated agents over specified time courses, cells were washed twice with ice-cold PBS, and reaction was terminated by addition of 100 µl of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 0.5 mM phenylmethyl sulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100). Protein (60 µg/lane) was separated on 7.5 to 12% SDS-polyacrylamide gel electrophoresis. The nitrocellulose membrane was immunoreacted with the primary antibody to ERK1/2, phosphorylated-ERK1/2 (BD Biosciences, San Jose, CA), Akt, phosphorylated-Akt, -tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and PKC (Serotec, Oxford, UK) for overnight incubation at 4°C. After four washings with PBS/0.1% Tween 20, the secondary antibody (diluted 1:2000) was applied to the membranes for 1 h at room temperature. Antibody-reactive bands were detected with an enhanced chemiluminescence kit (ECL; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK).
In Vivo Matrigel Plug Assay. Nude mice (6 weeks of age) were given subcutaneous injections of 500 µl of Matrigel (BD Biosciences) at 4°C with growth factor (150 ng/ml VEGF or bFGF). After injection, the Matrigel rapidly formed a plug. YC-1 (1–100 mg/kg/day) was given orally starting on day 1. After 7 days, the skin of the mouse was easily pulled back to expose the Matrigel plug, which remained intact. After quantitative differences were noted and photographed, hemoglobin was measured as an indication of blood vessel formation, using the Drabkin method (Drabkin reagent kit 525; Sigma-Aldrich, St. Louis, MO). The concentration of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel.
In Vivo Antitumor Activity Assay. A549 (1 x 107) cells in a volume of 100 µl were transplanted through the chest wall into the left pleural space of nude mice using a 26-gauge needle on day 0; a dose of 10 mg/kg/day YC-1 was given orally starting on day 1. Antitumor activity was assessed as the ratio of median survival time (MST) in the treatment group (T) to MST in the control group (C), and the results are shown as T/C: life span T/C (%) = (MST of drug-treated group/MST of control group) x 100. Long-term survivors were recorded until each animal died.
Cytotoxicity Assay. The cytotoxicity assay was carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. MTT (Sigma-Aldrich) was dissolved in PBS at a concentration of 5 mg/ml and sterilized by filtration (Millipore Corporation, Billerica, MA). From this stock solution, 10 µl/100 µl medium was added to each well, and plates were gently shaken and incubated at 37°C for 2 h. After the incubation period, cells were lysed with dimethyl sulfoxide and quantified at OD550 with an enzyme-linked immunosorbent assay reader.
Data Analysis and Statistics. Data are presented as mean ± S.E.M. or as percentage of control. Statistical comparisons between groups were performed using the Student's t test. P < 0.05 was considered statistically significant.
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Results |
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Effect of YC-1 on VEGF- and bFGF-Induced Cell Migration and Tube Formation of HUVECs. Next, we used well established assays to assess the potential regulatory role of YC-1 on cell migration and differentiation. YC-1 (3–30 µM) significantly inhibited, in a dose-dependent manner, VEGF- and bFGF-induced cell migration in a transwell assay (Fig. 2A). However, HUVECs treated with 10 ng/ml VEGF and bFGF reorganized and subsequently formed capillary-like structures (Fig. 2B). YC-1 (3–30 µM) caused a concentration-dependent blockage of the capillary tubes and did not inhibit cell viability (MTT assay). These results demonstrate that YC-1 has the ability to block VEGF- and bFGF-induced in vitro angiogenesis.
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). Therefore, we evaluated the possibility that the inhibitory effect of YC-1 might be mediated through its ability to interfere with VEGF- and bFGF-induced activation of these intracellular pathways. To determine whether YC-1 could modulate these active signaling pathways that are involved in cell functions, HUVECs were incubated with increasing amounts of YC-1 in vitro and analyzed for changes in activated Akt and ERK1/2. First, we assessed the effect of YC-1 (1 and 10 µM) on ERK1/2 phosphorylation (Fig. 3A). VEGF- and bFGF-induced ERK1/2 phosphorylation (10 ng/ml) was strongly inhibited by YC-1 in a concentration-dependent manner. Densitometric analysis indicated that VEGF- and bFGF-induced ERK1/2 phosphorylation was reduced by 20 to 40% in the presence of 1 µM YC-1 and by 50 to 60% in the presence of 10 µM YC-1 (Fig. 3B). Akt is a protein kinase recruited to the membrane and activated by phosphorylation as a direct consequence of PI3 kinase activity (Cantley, 2002). We next evaluated Akt phosphorylation levels to test whether YC-1 is also capable of inhibiting angiogenic growth factor-induced PI3-K activation. As shown in Fig. 3C, YC-1 (10 and 30 µM) significantly suppressed the phosphorylation of Akt in a concentration-dependent manner. Total Akt expression was not affected by YC-1 treatment. The ratio between phosphorylated Akt and total Akt was quantified for each of the points in this experiment, and the results are shown in Fig. 3D. These data suggest that YC-1 is able to block VEGF- and bFGF-induced ERK1/2 and Akt signaling pathways in endothelial cells.
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Effect of YC-1 on VEGF- and bFGF-Induced PKC Activation in HUVECs. In the previous study, YC-1 significantly inhibited VEGF- and bFGF-induced HUVEC proliferation. However, VEGF and bFGF seemto exert their mitogenic effects through activation of a PKC-dependent pathway in human endothelial cells (Kent et al., 1995; Wellner et al., 1999). We next evaluated whether YC-1 reduction in angiogenic activity is associated with reduced PKC activity. Cells were treated with YC-1 (0.1–10 µM) for 1 h followed by 10 ng/ml VEGF and bFGF for 30 min and 2 h, respectively. As shown in Fig. 4, YC-1 significantly suppressed VEGF- and bFGF-induced PKC translocation from cytosol to membrane in a concentration-dependent manner. These data suggest that YC-1 is able to block VEGF- and bFGF-induced PKC signaling pathways in endothelial cells.
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Discussion |
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). In the present study, we demonstrated that YC-1, an activator of soluble guanylyl cyclase (Ko et al., 1994), has antiangiogenic properties in vitro and in vivo that can suppress tumor growth. In an in vitro model system, we demonstrated that YC-1 markedly and dose dependently inhibited VEGF- and bFGF-induced endothelial cell proliferation, migration, and tube formation. VEGF and bFGF are major angiogenic factors associated with induction and maintenance of the neovasculature in tumors (Dow and DeVere, 2000; Karkkainen and Petrova, 2000). Half-maximal inhibition was <3 µM. However, under the same experimental conditions, YC-1 inhibition of the specific endothelial cell response to VEGF and bFGF was much stronger than inhibition of normal cell growth of endothelial cells. Previous studies demonstrated that YC-1 activates sGC through an NO-independent pathway (Ko et al., 1994; Yu et al., 1995) or through reducing the activity of phosphodiesterase (Friebe et al., 1998; Galle et al., 1999; Koglin et al., 2002) and that most of the actions of YC-1 are cGMP mediated. On the other hand, YC-1 can enhance the activation of sGC by NO or NO donors (Friebe et al., 1998; Rothermund et al., 2000; Friebe and Koesling, 2003). However, our experiments demonstrated that ODQ (an sGC inhibitor) and L-NAME (a nitric oxide synthase inhibitor) did not reverse YC-1-induced antiproliferation in HUVECs. Therefore, we suggest that YC-1 inhibits VEGF- and bFGF-induced endothelial cells functions by a mechanism that is neither cGMP-dependent nor NO-mediated.
In preliminarily studies, we demonstrated that treatment with high concentrations (>50 µM) of YC-1 results in growth inhibition of hepatocellular carcinoma cells (HA22T) (Wang et al., 2005) or apoptosis of prostate cancer cells (PC-3) and leukemia cancer cells (HL-60 and CCRF-CEM) in culture (data not shown). Thus, the growth-inhibitory action of YC-1 at low concentration is considered to be a more specific effect on endothelial cells that are stimulated by angiogenic growth factors. Therefore, YC-1 may possess novel molecular properties that interfere with common angiogenic signaling pathways triggered by growth factor stimulation in endothelial cells.
As noted above, the activation of ERK1/2, PI3-K and PKC pathways are required for the proliferative and migratory effects of VEGF and bFGF on endothelial cells (Cross and Claesson-Welsh, 2001). However, YC-1 significantly inhibits angiogenic factor-induced ERK1/2 and Akt activities as well as PKC translocation from cytosol to membrane compartments in a concentration-dependent manner. Furthermore, the previous study demonstrates possible molecular mechanisms by which YC-1, at concentrations between 5 and 50 µM, inhibits FBS-induced proliferation of human vascular endothelial cells in vitro (Hsu et al., 2003). It is possible that the effect is mediated through induction of a cyclin-dependent kinase inhibitor, p21 or p27, but YC-1 did not induce any significant changes in cyclins or CDKs. However, in this study, our data show that YC-1 (0.01–1 µM) did not affect HUVEC proliferation stimulated by FBS. Under the same experimental conditions, YC-1 inhibited the mitogenic activity of VEGF and bFGF, but not FBS in the same concentration range. Taken together, the data suggest that YC-1 is most likely to inhibit cell proliferation of HUVEC through VEGF- and bFGF-mediated signaling, including ERK1/2, Akt, and PKC pathways that are requisite for the angiogenic activities of VEGF and bFGF in endothelial cells.
Data from the in vivo Matrigel plug implantation animal model indicated that YC-1 markedly inhibited angiogenic growth factor-induced new vessel formation in a dose-dependent manner. Considering that angiogenesis is essential for tumor growth, the antitumor activity of YC-1 may be related to its antiangiogenic activities. Subsequently, we used the high angiogenic tumor A549, a nonsmall cell lung cancer, to assess YC-1 antitumor activity. We monitored body weights of mice every three dramatically loss from 20 g to 12 to 13 g. The results showed that YC-1 prolonged animal survival rate (>180%) after tumor cell implantation without producing cytotoxicity. However, Chun et al. (2001) and Yeo et al. (2003) demonstrated that YC-1 is an inhibitor of HIF-1, which halts tumor growth (by blocking tumor angiogenesis) and tumor adaptation to hypoxia. YC-1 (
5 x 10-6 M) suppressed HIF-1 protein level and expression of several HIF-1-regulated genes (VEGF, aldolase A, and enolase 1) in cancer cells under hypoxic conditions. YC-1 in our study showed efficacy in endothelial cells by inhibiting VEGF- and bFGF-induced angiogenic functions, such as proliferation (IC50 < 1 x 10-7 M), migration, and differentiation (both IC50 < 3 x 10-6 M), more than it inhibited the expression of HIF-1-regulated genes in cancer cells (ratio of 2- to 20-fold greater inhibition). Previous studies demonstrated that intraperitoneal injection of YC-1 (30 mg/kg) abrogates tumor growth and is associated with the suppression of tumor angiogenesis in animal models (Yeo et al., 2003). Furthermore, we showed that oral administration of YC-1 (ID50 = 3 mg/kg) inhibited VEGF- and bFGF-induced new vessel formation in the Matrigel plug implantation model. These antiangiogenic activities of YC-1 in vivo may be explained by their direct inhibition of angiogenic factor-induced cell proliferation, migration, and differentiation in endothelial cells. Taken together, we believe that YC-1 is worth investigating further for clinical applications in cancer therapy.
In conclusion, our results demonstrated that YC-1 inhibited angiogenic responses in vitro and in vivo by blocking angiogenic factor signaling pathways, such as ones involving ERK1/2, Akt, and PKC. Inhibition of tumor activity by YC-1 is related to its antiangiogenic activity, which correlates with blockage of VEGF- and bFGF-induced endothelial cell proliferation, migration, and tube formation. Taken together, our results suggest that YC-1 is a potent angiogenesis inhibitor with the potential to become a useful agent in the treatment of human cancer and other angiogenesis-dependent diseases.
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Footnotes |
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S.-L.P. and J.-H.G. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole; sGC, soluble guanylyl cyclase; HUVEC, human umbilical vascular endothelial cell; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PKC, protein kinase C; ERK1/2, extracellular signal-regulated protein kinase 1/2; MST, median survival time; ODQ, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one; L-NAME, NG-nitro-L-arginine methyl ester; HIF-1, hypoxia-inducible factor-1.
Address correspondence to: Dr. Che-Ming Teng, Pharmacological Institute, College of Medicine, National Taiwan University, 1 Jen-Ai Road, Section 1, Taipei, Taiwan. E-mail: cmteng{at}ha.mc.ntu.edu.tw
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