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Blocking S1P interaction with S1P1 receptor by a novel competitive S1P1-selective antagonist inhibits angiogenesis
Biochemical and Biophysical Research Communications 419 (2012) 754–760
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Blocking S1P interaction with S1P1 receptor by a novel competitive S1P1-selective antagonist inhibits angiogenesis Yasuyuki Fujii a,⇑, Yasuji Ueda a, Hidenori Ohtake a, Naoya Ono a, Tetsuo Takayama a, Kiyoshi Nakazawa a, Yasuyuki Igarashi b, Ryo Goitsuka c a b c
Department of Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co. Ltd., 1-403 Saitama, Saitama 331-9530, Japan Laboratory of Biomembrane and Biofunctional Chemistry, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan Division of Development and Aging, Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan
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Article history: Received 18 January 2012 Available online 24 February 2012 Keywords: Angiogenesis Antagonist Sphingosine 1-phosphate Type 1 S1P receptor Vascular endothelial growth factor
a b s t r a c t Sphingosine 1-phosphate receptor type 1 (S1P1) was shown to be essential for vascular maturation during embryonic development and it has been demonstrated that substantial crosstalk exists between S1P1 and other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. We developed a novel S1P1-selective antagonist, TASP0277308, which is structurally unrelated to S1P as well as previously described S1P1 antagonists. TASP0277308 inhibited S1P- as well as VEGF-induced cellular responses, including migration and proliferation of human umbilical vein endothelial cells. Furthermore, TASP0277308 effectively blocked a VEGF-induced tube formation in vitro and significantly suppressed tumor cell-induced angiogenesis in vivo. These findings revealed that S1P1 is a critical component of VEGF-related angiogenic responses and also provide evidence for the efficacy of TASP0277308 for anti-cancer therapies. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Angiogenesis is the process by which new blood vessels are formed from existing vasculature. During angiogenesis, migration and proliferation of vascular endothelial cells are important processes to maintain net vascular structure by replenishing new endothelial cells toward physiological and pathological stimuli, such as wound healing and tumor growth. Tumor angiogenesis is considered to be a crucial component of disease progression, and thus is one of potent pharmacological targets for anti-cancer therapy [1–3]. Sphingosine-1-phosphate (S1P), a potent lipid-mediator produced from the metabolism of sphingosine by sphingosine kinase 1 (SphK1) and SphK2, acts on a family of G protein-coupled receptors (S1P1–5), and transduces intracellular signals involved in numerous physiological and pathological cellular processes Abbreviations: S1P, sphingosine 1-phosphate; S1P1, the type 1 sphingosine 1phosphate (S1P) G protein-coupled receptor; Ab, antibody; HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum; BSA, bovine serum albumin; ECs, endothelial cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; NGF, nerve growth factor; TNF, tumor necrosis factor; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor. ⇑ Corresponding author. Address: 1-403 Yoshino-cho, Saitama-shi, Saitama 3319530, Japan. Fax: +81 48 669 7254. E-mail address: [email protected] (Y. Fujii). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.096
[4,5]. Three subtypes of S1P receptors, S1P1, S1P2 and S1P3, are expressed on vascular endothelial cells [6]; S1P1 couples stringently to the Gi protein family for signal transduction, whereas S1P2 and S1P3 couple to the Gi, Gq and G12/13 protein families [7]. Multiple interconnections of S1P signaling through S1P1 and S1P3 induce vascular endothelial cell proliferation, migration, morphogenesis, cytoskeletal reorganization, and adherens junction assembly, whereas S1P2 apparently transduces signals that negatively regulate S1P-mediated multiple responses of vascular endothelial cells [8]. The in vivo evidence supporting the contribution of S1P and S1P1 to tumor angiogenesis is recently demonstrated as neutralization of the action of S1P by the specific antibody or RNA interference-mediated S1P1 knock-down significantly inhibits angiogenesis, tumor growth and metastasis [9,10]. Many growth and pro-angiogenic factors that have been implicated in tumor angiogenesis including vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), have also been demonstrated to substantially crosstalk with S1P– S1P receptor signaling in angiogenesis [11–14]. However, it still remains to be unsolved of how the signaling pathways from different S1P receptors and also between S1P receptors and other pro-angiogenic factors are integrated in the process of tumor angiogenesis. We have recently developed a novel S1P1-seletive antagonist, TASP0277308 [15]. TASP0277308 competitively inhibits S1P bind-
Y. Fujii et al. / Biochemical and Biophysical Research Communications 419 (2012) 754–760
ing to S1P1, S1P-induced receptor internalization, and S1P-induced signaling downstream of S1P1, including GTPcS-binding and cAMP formation. TASP0251078 is unique in its chemical structure bearing a sulfonamide moiety and lacks the alkyl side chain, while others, including VPC03090, VPC23019, VPC44116 and W146, are alkyl phenyl amide phosphonates that have been generated as a series of structural analogs of FTY720-phosphate [16–19]. Furthermore, although previously-generated S1P-relative antagonists failed to induce lymphopenia, TASP0277308 has the in vivo activity for inducing lymphocyte sequestration from the peripheral blood, inhibiting thymic egress and relocalizing marginal zone B cells from marginal zones to the splenic follicles, all of which recapitulate loss of function phenotypes of S1P1-deficient mice. Chemical approaches to investigate the functions of S1P1 in angiogenesis have been performed by utilizing several S1P1 antagonists and agonists, however, these reagents were not selective for S1P1 [20–22], so that it is difficult to define these results to formally understand the role of S1P1 in angiogenesis. In the present study, we thus used a highly S1P1-selective antagonist, TASP0277308, as a chemical tool to competitively inhibit the interaction of S1P with S1P1 in vitro and in vivo models designed to understand the role of S1P1 in angiogenesis. 2. Materials and methods 2.1. Reagents TASP0277308 was synthesized in our laboratories, as previously described [15]. S1P was purchased from Biomol (PA, USA). Recombinant VEGF165 and recombinant bFGF were purchased form R&D System Inc. (MN, USA). [3H]-tymidine was purchased form GE Healthcare (Uppsala, Sweden). Pertussis toxin (PTX) was purchased form Sigma (MI, USA). 2.2. Cell culture Human umbilical vein endothelial cells (HUVECs), purchased from Cambrex (NJ, USA), were maintained in EBM-2 complete
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medium (Cambrex) at 37 °C in a humidified with 5% CO2, and cells from 3 to 10 passages were used for the experiments in this study. Human fibrosarcoma HT1080 cells, obtained from the American Type Culture Collection (VA, USA), were also maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics. 2.3. Migration assay S1P-induced migration assays were performed in 96-well transwell chambers with 8 lm polycarbonate membrane filters (Neuro Probe, MD, USA). After serum starvation in EBM-2 medium supplemented with 0.2% fetal bovine serum (hereafter referred to as SFEBM-2) for 30 min, cells were plated in triplicate into the top chamber at 5 104 cells/well in 65 ll SF-EBM-2 medium. The bottom chambers were loaded with SF-EBM-2 medium containing 100 ng/ml S1P in the absence or presence of the indicated concentrations of TASP0277308. SF-EBM-2 medium without S1P were used as controls. Cells were allowed to migrate for 4 h in a humidified chamber at 37 °C with 5% CO2, and then cells migrated into the bottom chambers were counted by using the ATPlite-M Luminescence Assay System (PerkinElmer Life Science, MA, USA). VEGF-induced cell migration assays were carried out using modified Boyden chambers consisting of Chemotaxicell (Kurabo Industries Ltd., Osaka, Japan) membrane having 8 lm pore size filter inserts in 24-well tissue culture plates. The lower surfaces of the transwell membranes were coated with human collagen type-I (Daiichi Fine chemicals, Toyama, Japan). Cells were starved for 24 h and plated into the top chamber at 1.5 105 cells/well in 333 ll SF-EBM-2 medium. VEGF165 was added to the lower compartment and the indicated amounts of TASP0277308 were also added to both top and lower compartments. After 8 h incubation, the filters were fixed, and cells that had migrated to the lower surface were stained with 0.5% crystal violet in 20% methanol for 5 min. After several washes, the cells remaining on the upper surface were removed with cotton bud. The stained cells to the lower surface were extracted with 30% acetic acid, and the absorbance was measured at 595 nm.
Fig. 1. TASP0277308 inhibits S1P- and VEGF-induced migration of HUVEC. (A) Chemical structures of TASP0277308 and S1P. (B and C) HUVEC were cultured with 30 nM S1P for 4 h (B) or 10 ng/ml VEGF for 24 h (C) in the presence or absence of the indicated concentration of TASP0277308. The number of cells migrated toward S1P or VEGF were counted, as described in Section 2. Each point represents the mean ± SD of triplicate determinations.
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Fig. 2. TASP0277308 inhibits cell proliferation of on HUVECs induced by S1P, VEGF or bFGF. Cells were treated with the indicated concentrations of TASP0277308 with 500 nM S1P (A), 30 ng/ml VEGF (B) or 3 ng/ml bFGF (C) for 24 h, and then cell proliferation was evaluated using the [3H]-thymidine incorporation, as described in Section 2. Each point represents the mean ± SD of triplicate determinations. (D) The cells pretreated (shaded bars) or untreated (open bars) with 100 ng/ml PTX for 6 h were cultured with the indicated concentration of bFGF, VEGF or S1P for 24 h, as described in Section 2. Cell proliferation was measured as in (A). ⁄p < 0.05, ⁄⁄p < 0.01 (Student’s t-test).
2.4. Proliferation assay Cell proliferation was assessed by [3H]-thymidine incorporation according to the protocol described by Lee et al. [23]. HUVECs were seeded at 3 103 cells/well on collagen-coated 96-well plates and allowed to attach overnight. After serum starvation in SF-EBM-2 medium for 6 h, the cells were incubated with S1P (500 nM), bFGF (3 ng/ml) or VEGF (30 ng/ml) in SF-EBM-2 medium with hydrocortisone and heparin for 24 h. The cells were treated with 1 lCi [3H]-thymidine/well 2 h before termination of the assay, and the radioactivity of incorporated [3H]-thymidine was determined in a liquid scintillation counter (PerkinElmer Life Science). Experiments were carried out in triplicates and repeated three times. 2.5. In vitro tube formation assay The tube formation of HUVECs was performed using an angiogenesis kit (Kurabo Industries Ltd), according to the manufacturer’s instruction. Briefly, HUVECs and fibroblasts were co-cultured with VEGF (10 ng/ml) in the absence or presence of various amounts of TASP0277308 for 11 days in the medium supplied from the kit. The medium containing VEGF and/or TASP0277308 was changed every 3 days. The wells were then fixed, stained with anti-CD31 antibody (Kurabo Industries Ltd), and were visualized according to the manufacturer’s protocol. The photographic images were acquired using a Nikon DIAPHOT-TMD microscope equipped with COOLPIX 4500 digital camera. The area covered by the tube network was quantitatively analyzed using Angiogenesis Image Analyzer (Kurabo Industries Ltd.). Analyses of the samples were
performed in triplicate and independent experiments were repeated three times. 2.6. Tumor angiogenesis The tumor-mediated angiogenesis was examined with HT1080 cells by using the mouse dorsal air-sac method [24] with a minor modification. An aliquot (0.15 ml) of HT1080 cell suspension (5 106 cell/ml) were injected into Millipore chamber consisting of Millipore filter ring (Millipore Co., MA, USA) and two Millipore filters (0.22 lm pore size). The chamber containing HT1080 cells was implanted into a dorsal air sac produced in an ICR mouse (7– 9 weeks, Charles River, Yokohama, Japan). HBSS-containing chamber was used as controls. Groups of eight mice were orally administered the indicated dosages of TASP0277308 twice a day for 5 days. After the implanted chamber was removed from the subcutaneous air sac fascia, a black Millipore ring was placed on the implanted site and the area encircled by the black ring was then photographed. The angiogenic response was assessed by counting the number of newly formed tortuous blood vessels of above 3 mm in length within the area encircled by the black ring. All animal studies were performed according to the guideline of the Japanese Experimental Animal Research Association, and were approved by Taisho Pharmaceutical Co., Ltd. Animals Care Committee. 2.7. Statistical analyses Statistical comparisons between groups were performed using the Student’s t-test, Welch’s t-test or parametric Dunnett’s test.
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Fig. 3. TASP0277308 blocks capillary-like tube formation of HUVECs. (A) Representative photographs of CD31-positive capillary-like structures of HUVECs. Cells were cultured without VEGF or with 10 ng/ml VEGF in the presence or absence of the indicated concentration of TASP0277308 for 11 days (n = 3). (B) Percentage of the tube formation following treatment with the indicated concentrations of TASP0277308 relative to the DMSO-treated control. The tube formation was judged using the software described in Section 2. Data represent mean ± SE of three independent experiments. #p < 0.001, significantly different from the non-treated group (Welch’s t-test), ⁄⁄⁄ p < 0.001, significantly different from DMSO-treated control (parametric Dunnett’s test).
3. Results and discussion 3.1. TASP0277308 inhibits S1P- and VEGF-induced migration of HUVECs TASP0251078 was originally identified as a functional S1P1 antagonist through the screening of chemical compounds selectively inhibiting the binding of S1P to HEK 293 cell line stably expressing human S1P1 [15]. TASP0277308 bears a sulfonamide moiety and lacks the alkyl side chain, thus structurally different from S1P (Fig. 1A), but has highly selective competitive antagonistic activity against S1P1, in terms of S1P binding and subsequent receptor internalization as well as S1P1-mediated signaling [15].
Since endothelial cell proliferation and migration are critical for angiogenesis, we first assessed the effect of TASP0277308 on HUVEC migration using an in vitro modified Boyden chamber method. HUVECs were seeded in the upper chamber and allowed to migrate into the lower chamber, which contained an indicated ligand (30 nM of S1P or 10 ng/ml VEGF) with or without TASP0277308. As shown in Fig. 1B and C, TASP0277308 was dose-dependently inhibited HUVEC migrated to the lower chamber in response to S1P and VEGF. The inhibitory activities of S1P- and VEGF-induced chemotaxis by TASP0277308 were estimated to be IC50 values of 1.6 ± 0.2 and 1.1 ± 0.4 nM, respectively. These activities of TASP0277308 on vascular endothelial cells are approximately similar to those for competitive ligand binding and other antagonistic
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Fig. 4. TASP0277308 inhibits tumor angiogenesis. (A) Representative views of vessel formation on the inner side of the dorsal skin of mice 5 days after implantation of a chamber filled with or without HT1080 cells. Mice implanted with a chamber containing HT1080 cells were treated orally with 10, 30, and 100 mg/kg of TASP0277308 twice a day for five consecutive days from implantation, respectively. Arrowheads indicate tortuous newly formed blood vessels of above 3 mm in length. (B) The number of newly formed vessels. Angiogenesis was judged using the index described in Section 2. Data represent the mean ± SE (n = 8) from three independent experiments. #p < 0.05 significantly different from the non-challenge group (Welch’s t-test), ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, significantly different from the untreated with TASP0277308 control group (parametric Dunnett’s test).
activities of TASP0277308 [15]. These findings thus demonstrated that S1P1 antagonism effectively inhibits vascular endothelial cell migration induced by VEGF as well as S1P. 3.2. TASP0277308 inhibits growth factor-induced proliferation of HUVECs We next examined the effect of TASP0277308 on vascular EC proliferation induced by bFGF, VEGF or S1P by measuring [3H]-thymidine incorporation into HUVEC. As similar to the effect of
TASP0277308 on vascular endothelial cell migration, TASP0277308 inhibited S1P- and VEGF-induced proliferation of HUVECs in dosedependent manners with IC50 values of 3.6 ± 0.1 and 3 ± 0.6 nM, respectively (Fig. 2A and B). Furthermore, bFGF-induced proliferation of HUVECs was also inhibited by TASP0277308 (Fig. 2C), although the antagonistic activity of TASP0277308 on bFGF-induced cell proliferation was substantially weaker (IC50 values of 775 ± 315 nM) than that on VEGF- or S1P-induced proliferation. Since HUVEC proliferation induced not only by S1P but also by VEGF and bFGF were completely blocked by the treatment with
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PTX, a Gi protein-specific inhibitor (Fig. 2D), the observed effects of TASP0277308 on endothelial cell proliferation appeared to be mediated by the inhibition of hetero-trimeric Gi protein signaling pathway downstream of S1P1.
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may be a promising therapeutic approach in the control of tumor metastasis as well as pathological angiogenesis. Continuing efforts to further improve this class of S1P1 antagonists will also be highly beneficial to develop anti-angiogenesis drugs for the therapy of various angiogenesis related disorders.
3.3. TASP0277308 inhibits VEGF-induced tube formation Acknowledgments We then examined the effect of TASP0277308 on tubular morphogenesis of vascular endothelial cells. When HUVECs were cocultured with human fibroblasts and incubated for 11 days with 10 ng/ml of VEGF, an increase in the number of tube-like structures was observed (Fig. 3A, upper left panel), whereas the HUVEC culture in the absence of VEGF exhibited only a few such structures (Fig. 3A, upper right panel). TASP0277308 was found to significantly inhibit the width and length of the endothelial tubes (Fig. 3A, lower panels). Counting of tube-like structure after immunostaining for CD31 revealed that the inhibitory effect of TASP0277308 on tube formation is maximal at the concentration of 2 lM, a dose at which the angiogenic effect of VEGF was completely canceled by TASP0277308 to the basal level of tube formation in the absence of VEGF (Fig. 3B). These results indicated that TASP0277308 has the ability to inhibit vascular morphogenesis by selectively blocking the interaction of S1P with S1P1. 3.4. TASP0277308 inhibits tumor angiogenesis in vivo We finally examined the efficacy of TASP0277308 on tumor angiogenesis by using the dorsal air sac model, in which angiogenesis is induced by subcutaneously implanted human fibrosarcoma HT1080 tumor cells that are shown to secrete high amounts of angiogenic factors such as VEGF [25]. Compared to the implanted controls without tumor cells (Fig. 4A, upper left panel), implantation of HT1080 cells clearly induced branching and morphogenesis of new vasculature in the mouse dorsal air sac (Fig. 4A, arrowheads in upper middle panel). In contrast, oral administration of TASP0277308 potently inhibited HT1080 cell-induced formation of new microvessels in a dose-dependent manner (Fig. 4A, lower panels). Counting of the number of newly formed blood vessels revealed that tumor angiogenesis was completely inhibited by oral administration of TASP0277308 (100 mg/ml) to the level comparable to implanted controls without tumor cells (Fig. 4B). TASP0277308 also inhibited tumor angiogenesis induced by mouse sarcoma S-180 cells (Y.F., unpublished data), which secrete VEGF [24]. Taken together, these results indicated that TASP0277308, via inhibiting the interaction of S1P with S1P1, effectively suppress tumor angiogenesis in vivo. 3.5. Concluding remarks Complex crosstalk between S1P and VEGF signaling pathways has been revealed to be involved in angiogenesis under both physiological and pathological conditions. For instance, it has been demonstrated that VEGF not only upregulates S1P1 expression on vascular endothelial cells [11] but also stimulates Sphk1 to produce S1P [26], resulting in the amplification of S1P/S1P1 signaling pathway in angiogenesis. On the other hand, S1P is also shown to increase VEGF production [27] as well as VEGF receptor-mediated ERK activation [28], leading to the activation of VEGF receptor-mediated angiogenesis. Based on our present findings that a highly selective S1P1 antagonist, TASP0277308, completely inhibits VEGF-induced migration, proliferation and morphogenesis of vascular endothelial cells both in vitro and in vivo, the angiogenesis induced by VEGF appeared to rely on the interaction of S1P with S1P1 expressed on vascular endothelial cells. Furthermore, since TASP0277308 effectively inhibited tumor-induced angiogenesis, the blockage of S1P–S1P1 interaction on the tumor vasculature
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Blocking S1P interaction with S1P1 receptor by a novel competitive S1P1-selective antagonist inhibits angiogenesis Yasuyuki Fujii a,⇑, Yasuji Ueda a, Hidenori Ohtake a, Naoya Ono a, Tetsuo Takayama a, Kiyoshi Nakazawa a, Yasuyuki Igarashi b, Ryo Goitsuka c a b c
Department of Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co. Ltd., 1-403 Saitama, Saitama 331-9530, Japan Laboratory of Biomembrane and Biofunctional Chemistry, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan Division of Development and Aging, Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan
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Article history: Received 18 January 2012 Available online 24 February 2012 Keywords: Angiogenesis Antagonist Sphingosine 1-phosphate Type 1 S1P receptor Vascular endothelial growth factor
a b s t r a c t Sphingosine 1-phosphate receptor type 1 (S1P1) was shown to be essential for vascular maturation during embryonic development and it has been demonstrated that substantial crosstalk exists between S1P1 and other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. We developed a novel S1P1-selective antagonist, TASP0277308, which is structurally unrelated to S1P as well as previously described S1P1 antagonists. TASP0277308 inhibited S1P- as well as VEGF-induced cellular responses, including migration and proliferation of human umbilical vein endothelial cells. Furthermore, TASP0277308 effectively blocked a VEGF-induced tube formation in vitro and significantly suppressed tumor cell-induced angiogenesis in vivo. These findings revealed that S1P1 is a critical component of VEGF-related angiogenic responses and also provide evidence for the efficacy of TASP0277308 for anti-cancer therapies. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Angiogenesis is the process by which new blood vessels are formed from existing vasculature. During angiogenesis, migration and proliferation of vascular endothelial cells are important processes to maintain net vascular structure by replenishing new endothelial cells toward physiological and pathological stimuli, such as wound healing and tumor growth. Tumor angiogenesis is considered to be a crucial component of disease progression, and thus is one of potent pharmacological targets for anti-cancer therapy [1–3]. Sphingosine-1-phosphate (S1P), a potent lipid-mediator produced from the metabolism of sphingosine by sphingosine kinase 1 (SphK1) and SphK2, acts on a family of G protein-coupled receptors (S1P1–5), and transduces intracellular signals involved in numerous physiological and pathological cellular processes Abbreviations: S1P, sphingosine 1-phosphate; S1P1, the type 1 sphingosine 1phosphate (S1P) G protein-coupled receptor; Ab, antibody; HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum; BSA, bovine serum albumin; ECs, endothelial cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; NGF, nerve growth factor; TNF, tumor necrosis factor; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor. ⇑ Corresponding author. Address: 1-403 Yoshino-cho, Saitama-shi, Saitama 3319530, Japan. Fax: +81 48 669 7254. E-mail address: [email protected] (Y. Fujii). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.096
[4,5]. Three subtypes of S1P receptors, S1P1, S1P2 and S1P3, are expressed on vascular endothelial cells [6]; S1P1 couples stringently to the Gi protein family for signal transduction, whereas S1P2 and S1P3 couple to the Gi, Gq and G12/13 protein families [7]. Multiple interconnections of S1P signaling through S1P1 and S1P3 induce vascular endothelial cell proliferation, migration, morphogenesis, cytoskeletal reorganization, and adherens junction assembly, whereas S1P2 apparently transduces signals that negatively regulate S1P-mediated multiple responses of vascular endothelial cells [8]. The in vivo evidence supporting the contribution of S1P and S1P1 to tumor angiogenesis is recently demonstrated as neutralization of the action of S1P by the specific antibody or RNA interference-mediated S1P1 knock-down significantly inhibits angiogenesis, tumor growth and metastasis [9,10]. Many growth and pro-angiogenic factors that have been implicated in tumor angiogenesis including vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), have also been demonstrated to substantially crosstalk with S1P– S1P receptor signaling in angiogenesis [11–14]. However, it still remains to be unsolved of how the signaling pathways from different S1P receptors and also between S1P receptors and other pro-angiogenic factors are integrated in the process of tumor angiogenesis. We have recently developed a novel S1P1-seletive antagonist, TASP0277308 [15]. TASP0277308 competitively inhibits S1P bind-
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ing to S1P1, S1P-induced receptor internalization, and S1P-induced signaling downstream of S1P1, including GTPcS-binding and cAMP formation. TASP0251078 is unique in its chemical structure bearing a sulfonamide moiety and lacks the alkyl side chain, while others, including VPC03090, VPC23019, VPC44116 and W146, are alkyl phenyl amide phosphonates that have been generated as a series of structural analogs of FTY720-phosphate [16–19]. Furthermore, although previously-generated S1P-relative antagonists failed to induce lymphopenia, TASP0277308 has the in vivo activity for inducing lymphocyte sequestration from the peripheral blood, inhibiting thymic egress and relocalizing marginal zone B cells from marginal zones to the splenic follicles, all of which recapitulate loss of function phenotypes of S1P1-deficient mice. Chemical approaches to investigate the functions of S1P1 in angiogenesis have been performed by utilizing several S1P1 antagonists and agonists, however, these reagents were not selective for S1P1 [20–22], so that it is difficult to define these results to formally understand the role of S1P1 in angiogenesis. In the present study, we thus used a highly S1P1-selective antagonist, TASP0277308, as a chemical tool to competitively inhibit the interaction of S1P with S1P1 in vitro and in vivo models designed to understand the role of S1P1 in angiogenesis. 2. Materials and methods 2.1. Reagents TASP0277308 was synthesized in our laboratories, as previously described [15]. S1P was purchased from Biomol (PA, USA). Recombinant VEGF165 and recombinant bFGF were purchased form R&D System Inc. (MN, USA). [3H]-tymidine was purchased form GE Healthcare (Uppsala, Sweden). Pertussis toxin (PTX) was purchased form Sigma (MI, USA). 2.2. Cell culture Human umbilical vein endothelial cells (HUVECs), purchased from Cambrex (NJ, USA), were maintained in EBM-2 complete
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medium (Cambrex) at 37 °C in a humidified with 5% CO2, and cells from 3 to 10 passages were used for the experiments in this study. Human fibrosarcoma HT1080 cells, obtained from the American Type Culture Collection (VA, USA), were also maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics. 2.3. Migration assay S1P-induced migration assays were performed in 96-well transwell chambers with 8 lm polycarbonate membrane filters (Neuro Probe, MD, USA). After serum starvation in EBM-2 medium supplemented with 0.2% fetal bovine serum (hereafter referred to as SFEBM-2) for 30 min, cells were plated in triplicate into the top chamber at 5 104 cells/well in 65 ll SF-EBM-2 medium. The bottom chambers were loaded with SF-EBM-2 medium containing 100 ng/ml S1P in the absence or presence of the indicated concentrations of TASP0277308. SF-EBM-2 medium without S1P were used as controls. Cells were allowed to migrate for 4 h in a humidified chamber at 37 °C with 5% CO2, and then cells migrated into the bottom chambers were counted by using the ATPlite-M Luminescence Assay System (PerkinElmer Life Science, MA, USA). VEGF-induced cell migration assays were carried out using modified Boyden chambers consisting of Chemotaxicell (Kurabo Industries Ltd., Osaka, Japan) membrane having 8 lm pore size filter inserts in 24-well tissue culture plates. The lower surfaces of the transwell membranes were coated with human collagen type-I (Daiichi Fine chemicals, Toyama, Japan). Cells were starved for 24 h and plated into the top chamber at 1.5 105 cells/well in 333 ll SF-EBM-2 medium. VEGF165 was added to the lower compartment and the indicated amounts of TASP0277308 were also added to both top and lower compartments. After 8 h incubation, the filters were fixed, and cells that had migrated to the lower surface were stained with 0.5% crystal violet in 20% methanol for 5 min. After several washes, the cells remaining on the upper surface were removed with cotton bud. The stained cells to the lower surface were extracted with 30% acetic acid, and the absorbance was measured at 595 nm.
Fig. 1. TASP0277308 inhibits S1P- and VEGF-induced migration of HUVEC. (A) Chemical structures of TASP0277308 and S1P. (B and C) HUVEC were cultured with 30 nM S1P for 4 h (B) or 10 ng/ml VEGF for 24 h (C) in the presence or absence of the indicated concentration of TASP0277308. The number of cells migrated toward S1P or VEGF were counted, as described in Section 2. Each point represents the mean ± SD of triplicate determinations.
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Fig. 2. TASP0277308 inhibits cell proliferation of on HUVECs induced by S1P, VEGF or bFGF. Cells were treated with the indicated concentrations of TASP0277308 with 500 nM S1P (A), 30 ng/ml VEGF (B) or 3 ng/ml bFGF (C) for 24 h, and then cell proliferation was evaluated using the [3H]-thymidine incorporation, as described in Section 2. Each point represents the mean ± SD of triplicate determinations. (D) The cells pretreated (shaded bars) or untreated (open bars) with 100 ng/ml PTX for 6 h were cultured with the indicated concentration of bFGF, VEGF or S1P for 24 h, as described in Section 2. Cell proliferation was measured as in (A). ⁄p < 0.05, ⁄⁄p < 0.01 (Student’s t-test).
2.4. Proliferation assay Cell proliferation was assessed by [3H]-thymidine incorporation according to the protocol described by Lee et al. [23]. HUVECs were seeded at 3 103 cells/well on collagen-coated 96-well plates and allowed to attach overnight. After serum starvation in SF-EBM-2 medium for 6 h, the cells were incubated with S1P (500 nM), bFGF (3 ng/ml) or VEGF (30 ng/ml) in SF-EBM-2 medium with hydrocortisone and heparin for 24 h. The cells were treated with 1 lCi [3H]-thymidine/well 2 h before termination of the assay, and the radioactivity of incorporated [3H]-thymidine was determined in a liquid scintillation counter (PerkinElmer Life Science). Experiments were carried out in triplicates and repeated three times. 2.5. In vitro tube formation assay The tube formation of HUVECs was performed using an angiogenesis kit (Kurabo Industries Ltd), according to the manufacturer’s instruction. Briefly, HUVECs and fibroblasts were co-cultured with VEGF (10 ng/ml) in the absence or presence of various amounts of TASP0277308 for 11 days in the medium supplied from the kit. The medium containing VEGF and/or TASP0277308 was changed every 3 days. The wells were then fixed, stained with anti-CD31 antibody (Kurabo Industries Ltd), and were visualized according to the manufacturer’s protocol. The photographic images were acquired using a Nikon DIAPHOT-TMD microscope equipped with COOLPIX 4500 digital camera. The area covered by the tube network was quantitatively analyzed using Angiogenesis Image Analyzer (Kurabo Industries Ltd.). Analyses of the samples were
performed in triplicate and independent experiments were repeated three times. 2.6. Tumor angiogenesis The tumor-mediated angiogenesis was examined with HT1080 cells by using the mouse dorsal air-sac method [24] with a minor modification. An aliquot (0.15 ml) of HT1080 cell suspension (5 106 cell/ml) were injected into Millipore chamber consisting of Millipore filter ring (Millipore Co., MA, USA) and two Millipore filters (0.22 lm pore size). The chamber containing HT1080 cells was implanted into a dorsal air sac produced in an ICR mouse (7– 9 weeks, Charles River, Yokohama, Japan). HBSS-containing chamber was used as controls. Groups of eight mice were orally administered the indicated dosages of TASP0277308 twice a day for 5 days. After the implanted chamber was removed from the subcutaneous air sac fascia, a black Millipore ring was placed on the implanted site and the area encircled by the black ring was then photographed. The angiogenic response was assessed by counting the number of newly formed tortuous blood vessels of above 3 mm in length within the area encircled by the black ring. All animal studies were performed according to the guideline of the Japanese Experimental Animal Research Association, and were approved by Taisho Pharmaceutical Co., Ltd. Animals Care Committee. 2.7. Statistical analyses Statistical comparisons between groups were performed using the Student’s t-test, Welch’s t-test or parametric Dunnett’s test.
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Fig. 3. TASP0277308 blocks capillary-like tube formation of HUVECs. (A) Representative photographs of CD31-positive capillary-like structures of HUVECs. Cells were cultured without VEGF or with 10 ng/ml VEGF in the presence or absence of the indicated concentration of TASP0277308 for 11 days (n = 3). (B) Percentage of the tube formation following treatment with the indicated concentrations of TASP0277308 relative to the DMSO-treated control. The tube formation was judged using the software described in Section 2. Data represent mean ± SE of three independent experiments. #p < 0.001, significantly different from the non-treated group (Welch’s t-test), ⁄⁄⁄ p < 0.001, significantly different from DMSO-treated control (parametric Dunnett’s test).
3. Results and discussion 3.1. TASP0277308 inhibits S1P- and VEGF-induced migration of HUVECs TASP0251078 was originally identified as a functional S1P1 antagonist through the screening of chemical compounds selectively inhibiting the binding of S1P to HEK 293 cell line stably expressing human S1P1 [15]. TASP0277308 bears a sulfonamide moiety and lacks the alkyl side chain, thus structurally different from S1P (Fig. 1A), but has highly selective competitive antagonistic activity against S1P1, in terms of S1P binding and subsequent receptor internalization as well as S1P1-mediated signaling [15].
Since endothelial cell proliferation and migration are critical for angiogenesis, we first assessed the effect of TASP0277308 on HUVEC migration using an in vitro modified Boyden chamber method. HUVECs were seeded in the upper chamber and allowed to migrate into the lower chamber, which contained an indicated ligand (30 nM of S1P or 10 ng/ml VEGF) with or without TASP0277308. As shown in Fig. 1B and C, TASP0277308 was dose-dependently inhibited HUVEC migrated to the lower chamber in response to S1P and VEGF. The inhibitory activities of S1P- and VEGF-induced chemotaxis by TASP0277308 were estimated to be IC50 values of 1.6 ± 0.2 and 1.1 ± 0.4 nM, respectively. These activities of TASP0277308 on vascular endothelial cells are approximately similar to those for competitive ligand binding and other antagonistic
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Fig. 4. TASP0277308 inhibits tumor angiogenesis. (A) Representative views of vessel formation on the inner side of the dorsal skin of mice 5 days after implantation of a chamber filled with or without HT1080 cells. Mice implanted with a chamber containing HT1080 cells were treated orally with 10, 30, and 100 mg/kg of TASP0277308 twice a day for five consecutive days from implantation, respectively. Arrowheads indicate tortuous newly formed blood vessels of above 3 mm in length. (B) The number of newly formed vessels. Angiogenesis was judged using the index described in Section 2. Data represent the mean ± SE (n = 8) from three independent experiments. #p < 0.05 significantly different from the non-challenge group (Welch’s t-test), ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, significantly different from the untreated with TASP0277308 control group (parametric Dunnett’s test).
activities of TASP0277308 [15]. These findings thus demonstrated that S1P1 antagonism effectively inhibits vascular endothelial cell migration induced by VEGF as well as S1P. 3.2. TASP0277308 inhibits growth factor-induced proliferation of HUVECs We next examined the effect of TASP0277308 on vascular EC proliferation induced by bFGF, VEGF or S1P by measuring [3H]-thymidine incorporation into HUVEC. As similar to the effect of
TASP0277308 on vascular endothelial cell migration, TASP0277308 inhibited S1P- and VEGF-induced proliferation of HUVECs in dosedependent manners with IC50 values of 3.6 ± 0.1 and 3 ± 0.6 nM, respectively (Fig. 2A and B). Furthermore, bFGF-induced proliferation of HUVECs was also inhibited by TASP0277308 (Fig. 2C), although the antagonistic activity of TASP0277308 on bFGF-induced cell proliferation was substantially weaker (IC50 values of 775 ± 315 nM) than that on VEGF- or S1P-induced proliferation. Since HUVEC proliferation induced not only by S1P but also by VEGF and bFGF were completely blocked by the treatment with
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PTX, a Gi protein-specific inhibitor (Fig. 2D), the observed effects of TASP0277308 on endothelial cell proliferation appeared to be mediated by the inhibition of hetero-trimeric Gi protein signaling pathway downstream of S1P1.
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may be a promising therapeutic approach in the control of tumor metastasis as well as pathological angiogenesis. Continuing efforts to further improve this class of S1P1 antagonists will also be highly beneficial to develop anti-angiogenesis drugs for the therapy of various angiogenesis related disorders.
3.3. TASP0277308 inhibits VEGF-induced tube formation Acknowledgments We then examined the effect of TASP0277308 on tubular morphogenesis of vascular endothelial cells. When HUVECs were cocultured with human fibroblasts and incubated for 11 days with 10 ng/ml of VEGF, an increase in the number of tube-like structures was observed (Fig. 3A, upper left panel), whereas the HUVEC culture in the absence of VEGF exhibited only a few such structures (Fig. 3A, upper right panel). TASP0277308 was found to significantly inhibit the width and length of the endothelial tubes (Fig. 3A, lower panels). Counting of tube-like structure after immunostaining for CD31 revealed that the inhibitory effect of TASP0277308 on tube formation is maximal at the concentration of 2 lM, a dose at which the angiogenic effect of VEGF was completely canceled by TASP0277308 to the basal level of tube formation in the absence of VEGF (Fig. 3B). These results indicated that TASP0277308 has the ability to inhibit vascular morphogenesis by selectively blocking the interaction of S1P with S1P1. 3.4. TASP0277308 inhibits tumor angiogenesis in vivo We finally examined the efficacy of TASP0277308 on tumor angiogenesis by using the dorsal air sac model, in which angiogenesis is induced by subcutaneously implanted human fibrosarcoma HT1080 tumor cells that are shown to secrete high amounts of angiogenic factors such as VEGF [25]. Compared to the implanted controls without tumor cells (Fig. 4A, upper left panel), implantation of HT1080 cells clearly induced branching and morphogenesis of new vasculature in the mouse dorsal air sac (Fig. 4A, arrowheads in upper middle panel). In contrast, oral administration of TASP0277308 potently inhibited HT1080 cell-induced formation of new microvessels in a dose-dependent manner (Fig. 4A, lower panels). Counting of the number of newly formed blood vessels revealed that tumor angiogenesis was completely inhibited by oral administration of TASP0277308 (100 mg/ml) to the level comparable to implanted controls without tumor cells (Fig. 4B). TASP0277308 also inhibited tumor angiogenesis induced by mouse sarcoma S-180 cells (Y.F., unpublished data), which secrete VEGF [24]. Taken together, these results indicated that TASP0277308, via inhibiting the interaction of S1P with S1P1, effectively suppress tumor angiogenesis in vivo. 3.5. Concluding remarks Complex crosstalk between S1P and VEGF signaling pathways has been revealed to be involved in angiogenesis under both physiological and pathological conditions. For instance, it has been demonstrated that VEGF not only upregulates S1P1 expression on vascular endothelial cells [11] but also stimulates Sphk1 to produce S1P [26], resulting in the amplification of S1P/S1P1 signaling pathway in angiogenesis. On the other hand, S1P is also shown to increase VEGF production [27] as well as VEGF receptor-mediated ERK activation [28], leading to the activation of VEGF receptor-mediated angiogenesis. Based on our present findings that a highly selective S1P1 antagonist, TASP0277308, completely inhibits VEGF-induced migration, proliferation and morphogenesis of vascular endothelial cells both in vitro and in vivo, the angiogenesis induced by VEGF appeared to rely on the interaction of S1P with S1P1 expressed on vascular endothelial cells. Furthermore, since TASP0277308 effectively inhibited tumor-induced angiogenesis, the blockage of S1P–S1P1 interaction on the tumor vasculature
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