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Experimental Therapeutics |
2 Subunit on Endothelium
Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan 300-2635
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2, 3, 5, and ß1 in confluent culture of HUVEC, and integrin 2 was initially suppressed in mRNA level, followed by decrement of integrins 3, 5, and ß1. The inhibition of integrin 2 expression in HUVEC showed dose dependence but did not alter the level of CD31. Up-regulation of integrin 2 by phorbol 12-myristate 13-acetate abrogated the inhibitory effect of E7820 on tube formation within type I collagen gel, whereas addition of antibody against integrin 2 canceled the phorbol 12-myristate 13-acetate effect. These results suggest that E7820 inhibited tube formation through the suppression of integrin 2. Oral administration of E7820 remarkably resulted in inhibition of tumor-induced angiogenesis in mouse dorsal air sac model, and tumor growth of human colorectal tumor cell lines (WiDr and LoVo) was inhibited in xenotransplanted model in mice. This is the first time that a small molecule has been shown to modulate integrins, and this finding may provide the basis for a new approach to antiangiogenic therapy through the suppression of integrin 2 on endothelium. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 induced endothelial cell apoptosis of newly formed vessels (12)
. Angiostatin, a fragment of plasminogen, and endostatin, a COOH-terminal fragment of collagen XVIII, showed antitumor activity in preclinical studies (13
, 14)
. In particular, endostatin caused tumor regression without inducing drug resistance in mouse models (15)
. These two inhibitors appear to act on integrin vß3 and 5, respectively (16
, 17)
, although their action mechanisms remain unclear, and they are currently under clinical study.
Aromatic sulfonamide derivatives exhibit a range of bioactivities, including antimicrobial (18)
, antidiabetic (19)
, anti-inflammatory (20)
, and anticancer (21
, 22
, 23)
. We speculated that a novel angiogenesis inhibitor might be found among sulfonamide derivatives and used a TF model for screening assay. The results led to the discovery of a novel antiangiogenic sulfonamide derivative, E7820, which modulates the expression of integrin 2, 3, 5, and ß1 on HUVEC. In the present study, we examined the action of E7820 on integrin subunits in a TF model and found that suppression of integrin 2 by E7820 played a crucial role in inhibition of endothelium TF. Moreover, E7820 was confirmed to show inhibition of tumor-induced angiogenesis and tumor growth in a xenotransplanted model in mice.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The chemical identity of E7820 was supported by nuclear magnetic resonance, mass spectroscopy, and elemental analysis. The chemical identity of TNP-470, SU5416, and marimastat was supported by nuclear magnetic resonance and mass spectroscopy data.
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Monoclonal Antibody.
P1B5 (human 3), JB1a (human ß1), LM609 (human Vß3), and P1F6 (human Vß5) were purchased from Chemicon International Inc. (Temecula, CA). 5E8D9 (human 1 subunit) and A2-IIE10 (human 2 subunit) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). SG/73 (human 4 subunit) and KH/33 (human 5 subunit) were purchased from Seikagaku Corporation (Tokyo, Japan). JC/70A (human CD31) and FITC-conjugated F(ab)2 fragment of rabbit antimouse immunoglobulins were purchased from Dako (Glostrup, Denmark). K20 (human ß1 subunit) was purchased from Immunotech (Marseilles, France). 4F10 (human 6 subunit) was purchased from Serotec (Sapporo, Japan). Rat monoclonal antimouse CD31 antibody (clone MEC13.3) was purchased from PharMingen (San Diego, CA).
Sandwich TF Assay.
Four-hundred µl aliquots of collagen gel were added to 24-well plates and allowed to gel for at least 1 h at 37°C. After gelation, HUVEC were plated on the gel at a concentration of 1–1.2 x 105 cells in SFM (Human endothelial-SFM Basal Growth Medium; Life Technologies, Inc., Grand Island, NY) with epidermal growth factor (Life Technologies, Inc.) at 10 ng/ml and either bFGF (Life Technologies, Inc.) or VEGF (Wako Pure Chemical Industries, Osaka, Japan) at 20 ng/ml. After culture at 37°C overnight, HUVEC were covered with 400 µl of similar collagen gel and left for at least 3 h at 37°C. SFM (1.5 ml) supplemented with epidermal growth factor and either bFGF or VEGF was added after gelation, with or without inhibitors. In the case of addition of antibody, 96-well plates and one-fourth amounts of reagents and cells were used. Tube length of capillaries was quantified by tracing the center of each formed tube and calculating the pixel intensity of the outlined image using Mac SCOPE 2.56 (Mitani Corporation, Chiba, Japan). All of the experiments were done at least in duplicate and repeated three times.
Cell Growth Assay.
HUVEC were grown in EGM kit (Sanko Junyaku, Tokyo, Japan) containing 2% FBS and plated at 800 cells/well onto 96-well plates in 0.1 ml of EGM kit containing 10% FBS. After 24 h, inhibitors or vehicle were added to duplicate cultures of cells and at 3 days after addition of inhibitors, the ratios of surviving cells were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The serum-free assays were carried out at 20,000 cells/well onto 24-well plates in 1 ml of human endothelial-SFM basal growth medium containing bFGF (20 ng/ml) or VEGF (20 ng/ml). All of the experiments were done at least in duplicate and repeated three times.
Analysis by Flow Cytometry.
Cells were harvested and suspended at 2–5 x 105 cells in 100 µl of PBS containing 0.1% BSA and 0.05% NaN3, then incubated with 1 µg of primary antibodies for 30 min at 4°C. Cells were washed with PBS, then incubated in 100 µl of FITC-conjugated secondary antibody diluted 1:50 in PBS for 30 min at 4°C, washed with PBS three times and fixed with CellFix (Becton Dickinson, Franklin Lakes, NJ). The control sample (for background) was incubated in PBS containing 0.1% BSA and 0.05% NaN3 without primary antibody. Fluorescence signals from 2 x 104 cells were acquired using a FACScalibur (Becton Dickinson, Mountain View, CA) to quantify staining intensity. The expression of each molecule was calculated using the mean fluorescence of each sample as determined by flow cytometry: relative expression (relative mean fluorescence intensity) = mean fluorescence intensity of sample/mean fluorescence intensity of background.
Mouse DAS Model.
The mouse DAS method was performed according to Sakamoto et al. (25)
with a minor modification (26)
. Briefly, a suspension of 1.5 x 107 cells in collagen gel was injected into a Millipore chamber consisting of a Millipore filter ring (PR0001401) and two Durapore filters (HVLP04700, 0.45 µm), which were attached to the ring with MF cement (XX7000000). This chamber was implanted into a DAS produced in C57BL/6 mice by injecting 10 ml of air through a 25-gauge needle.
Xenograft Model.
Six-week-old female nude mice (KSN mice) underwent s.c. transplantation of human tumors. Administration was started (day 1) at 3 days (WiDr cells) or 7 days (Lovo cells) after transplantation. E7820 was p.o. administered on a schedule of twice daily every day. The tumor volumes were followed during the experiment by direct measurement of the diameter of tumors with calipers, according to the formula: tumor volume = (a x b x b)/2, where a is the largest diameter and b is the diameter perpendicular to a. 
T/C (% of control for growth) were calculated from the formula; (T/C) x 100. T and C are changes in tumor volume (growth) for each treated and vehicle control group. In the case of reduction of tumor volume, T/C values were calculated following formula: T/C (%) = (TVn - TV1)/TV1 x 100, where TVn is the tumor volume of treated mice on day n.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inhibited both bFGF- and VEGF-driven TF of HUVEC in a dose-dependent manner (Fig. 1, b–d)
. The capillary network was broken up and changed to small islands. TNP-470, an inhibitor of endothelial cell proliferation (27)
, did not inhibit TF at 2.5 µg/ml (Fig. 1b)
, and SU-5416, an inhibitor of VEGF receptor kinase (28)
, inhibited only VEGF-driven TF (Fig. 1c
; Table 1
). Marimastat, a matrix metalloproteinase inhibitor (29)
, induced a slight morphological change of the capillary-tube network, but the tube number was not decreased (Fig. 1b)
.
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. The IC50 values were 0.10 and 0.081 µg/ml, respectively. E7820 showed similar inhibition of proliferation stimulated by bovine brain extract in medium containing 10% FBS. TNP-470 potently inhibited proliferation of HUVEC stimulated by bovine brain extract in medium containing 10% FBS, and SU-5416 inhibited proliferation of HUVEC stimulated by VEGF in SFM. Marimastat did not inhibit proliferation of HUVEC. However, all of the inhibitors (E7820, TNP-470, SU-5416, and marimastat) showed potent inhibitory activity in a rat aortic angiogenic model (30)
, which involves several angiogenic processes such as degradation of ECM, migration, proliferation, and TF. The activity profile shown in Table 1
indicates that E7820 has a unique character compared with the other angiogenesis inhibitors.
Effect of E7820 on Expression Levels of Integrin Subunits on Endothelium.
In the TF model, E7820 inhibited TF after the second collagen gel had been overlaid, whereas E7820 did not decrease the number of HUVEC on the first collagen gel at confluence (data not shown), suggesting that E7820 inhibits the TF process. Because cell-cell and/or cell-ECM adhesion molecules play an important role in the TF process, we firstly studied adhesion molecules on HUVEC to clarify the action mechanism of E7820. Flow cytometry analysis of HUVEC revealed that integrin subunits 2, 5, 6, and ß1 were highly expressed on the cell surface, but integrin 1 and 4 were not. The expression levels of integrin 2, 5, and 6 subunits were significantly down-regulated in confluent culture compared with subconfluent culture (Fig. 2a)
.
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2 was significantly decreased to 
50% of the control under both culture conditions (Fig. 2, b and c)
. Integrins 3 and 5 were also decreased, although to lesser extents than 2 subunit. The expression level of 6 subunit was significantly down-regulated to 60% of the control in subconfluent culture, but it remained at 90% of the control in confluent culture. E7820 treatment did not alter the expression of v integrin or CD31 under these culture conditions. TNP-470 at 0.05 µg/ml did not affect the pattern of integrin expression under the confluent culture condition (data not shown).
To investigate the decrease of integrins 2, 3, 5, and ß1 in more detail, we analyzed the alteration of the mRNAs by a quantitative PCR method. After 12 h treatment of HUVEC with E7820 (0.05 µg/ml), integrin 2 mRNA was significantly decreased, whereas other integrin subunits including CD31 and VE-cadherin were not altered (Fig. 3a)
. E7820 suppressed integrin 2 mRNA after 6 h of treatment, indicating that E7820 acts initially on integrin 2 followed by integrins 3, 5, and ß1 (Fig. 3b)
. The inhibition of integrin 2 expression in HUVEC showed dose dependence, but E7820 did not alter the level of CD31 even at the concentration of 0.5 µg/ml (Fig. 4)
. Furthermore, because the activity of E7820 toward integrin 2 was not observed in a human fibroblast cell line (WI-38; data not shown), the inhibitory action of E7820 might be selective to certain cell types.
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2 in the TF Model and Effect of E7820.2 antibody inhibited bFGF-driven TF of HUVEC in a dose-dependent manner (Fig. 5, a–c and e)
, but anti-5 antibody had no effect at the concentration of 20 µg/ml (Fig. 5d)
. Because the anti-2 antibody did not induce any change of HUVEC at confluent monolayer on the first collagen gel without overlaying the second gel, integrin 2 appears to play an important role in the TF process. Anti-integrin ß1 (a counterpart of integrin 2) antibody also inhibited TF of HUVEC. Similar results were obtained with anti-2 and -ß1 antibodies in VEGF-driven TF (data not shown).
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2 subunit is up-regulated through the activation of protein kinase C by PMA in some epithelial cells (31)
. The expression level of integrin 2 subunit on HUVEC was remarkably increased by treatment with 2 nM PMA for 24 h. However, integrin 3 and CD31 showed no change, although expression of integrin 5 was slightly stimulated (Fig. 6a)
. We next investigated whether PMA affected the decrease of integrin 2 expression on HUVEC by E7820. When HUVEC was treated with E7820 at 0.5 µg/ml, the expression level of integrin 2 was decreased. However, in the presence of 2 nM PMA, the expression level of integrin 2 recovered to the control level (Fig. 6b)
. The inhibitory activity of E7820 in the VEGF-driven TF model was also reduced by PMA in a dose-dependent manner (Fig. 6c)
. Inhibitory activity was recovered by addition of antibody against integrin 2, indicating that the reversing effect of PMA on the inhibition by E7820 is indeed because of stimulation of integrin 2 expression (Fig. 6d)
. A similar effect of PMA on E7820-treated HUVEC was also observed in bFGF-driven TF (data not shown).
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. Immunohistochemical analysis showed that newly formed vessels appeared beneath the muscle layer in the skin, whereas such a vessel-enriched layer was not found in mice given 400 mg/kg E7820 (Fig. 7b)
. Because WiDr cells are extremely less sensitive toward E7820 in cell growth assay (see below), the effect in DAS model strongly suggests an antiangiogenic potency of E7820. Moreover, E7820 (oral administration twice daily for 14 days) significantly delayed the growth of WiDr cells inoculated s.c. The antitumor effect was dose-dependent at 50, 100, and 200 mg/kg, and the tumor growth rates after the beginning of E7820 treatment (T/C value) were 52%, 46%, and 27%, respectively (Fig. 8a)
. E7820 also completely suppressed the growth of human colorectal tumor, LoVo cells, on the same administration schedule for 21 days at the doses of 100 and 200 mg/kg (Fig. 8b)
. Antiproliferative activity of E7820 in vitro against both WiDr and LoVo cells was very weak compared with that against HUVEC. The values of IC50 were 29 and 15 µg/ml, respectively, suggesting that the antitumor effects of E7820 resulted from antiangiogenic activity.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 was crucial for TF within type I collagen gel, although E7820 suppressed expression of integrins 2, 3, 5, and ß1 in confluent culture. Up-regulation of integrin 2 through the activation of PKC by PMA suppressed the effect of E7820 in the TF model, whereas addition of antibody against integrin 2 canceled the PMA effect, leading to inhibition of TF. These results suggest that PMA abrogates the inhibitory activity of E7820 on TF through increasing the expression of integrin 2. It is reported that the expression of both integrin 1 and 2 is regulated by VEGF in microvascular endothelial cells, and a combination of inhibitory antibodies against integrins 1 and 2 inhibited VEGF-driven angiogenesis in a Matrigel model in mice (32)
. Recently, it was reported that an inhibitory antibody against integrin 2 also inhibited tumor angiogenesis in a mouse model (33)
. Because the expression of integrin 1 was not detected in HUVEC, the suppression of integrin 2 seems to be sufficient for the inhibition of TF. Furthermore, Davis and Camarillo (34)
reported that vacuole and lumen formation within collagen gel are dependent on integrin 2, supporting our results.
The effect of E7820 on the expression of integrin 2 in HUVEC was detected at the mRNA level at 6 h after treatment. Because no alteration of the expression of integrin mRNAs, except integrin 2 mRNA, was detected at 12 h, the decrement of integrin 3, 5, and ß1 on the cell surface might be a secondary effect arising from the decrement of integrin 2 subunit. Because it has been reported that transfection of antisense DNA to integrin 2 mRNA led to a decrease of integrin ß1 on the cell surface (35)
, the alteration of integrin ß1 by E7820 may also result from the decrement of integrin 2 mRNA.
Inhibition rate of E7820 against integrin subunits was approximately up to 50% in HUVEC, whereas TF of HUVEC was clearly inhibited in collagen gel model. A similar phenomenon was reported by using antisense or DNAzyme to integrin ß1 and ß3 mRNA (36) . DNAzyme suppressed expression of integrin ß1 or ß3 mRNA by 40–50% in HUVEC; however, capillary TF in Matrigel completely inhibited, coinciding with our finding. These findings suggest that alternation of integrin expression on endothelial cells strongly affects capillary TF.
E7820 modulates integrin 2 mRNA in HUVEC, although its mechanism is unclear. Two possibilities can be considered. An analytical study of transcriptional regulation of integrin 2 was reported in megakaryotic cell lines, and it was speculated that the transcription of integrin 2 was regulated through the specific enhancer region of the 2 gene in platelets (37
, 38)
. Because E7820 showed different activities on integrin 2 expression in endothelial cells and fibroblast cells (data not shown), E7820 may act on a regulating molecule on an enhancer region in endothelial cells. Another possibility relates to integrin 2 mRNA stability. It was reported that ECM and growth factor affected the stability of mRNAs of integrin subunits. The stability of integrin v and ß3 mRNAs in endothelial cells was enhanced by fibrin compared with collagen (39)
, and platelet-derived growth factor increased the stability of integrin 2 mRNA in fibroblast cells cultured in collagen gel, whereas on tissue culture plates, platelet-derived growth factor stabilized integrin 3 and 5 mRNAs but not 2 mRNA (40)
. Although the mechanism of regulation of integrin 2 mRNA stability is not reported in endothelial cells, E7820 may act on this modulation system of integrin mRNAs. Our preliminary data suggests that E7820 destabilizes integrin 2 mRNA in HUVEC.
Integrin consists of heterodimers of 18 subunits and 8 ß subunits. It was reported that antagonists to integrin vß3 or vß5 induced apoptosis of endothelium in newly formed vessels (41)
, and inhibited in vivo angiogenesis and tumor growth, although E7820 did not affect the expression of v. However, observations in integrin v-, ß3-, and ß3/ß5-null mice seemed inconsistent with the above findings. All of the null mice exhibited angiogenesis, and furthermore, ß3/ß5-null mice showed enhanced-tumor growth (42
, 43)
. These results suggest that antagonism and suppression of integrin expression result in different effects depending on the integrin subunits involved. Integrin ß1 was also thought to be necessary for tumor-induced angiogenesis. Although ES cells formed large teratomas, including microvessels differentiated from the ES cells, after inoculation into nude mice, integrin ß1 null cells grew only small tumors, which did not contain microvessels derived from ES cells. Furthermore, studies of knockout mice revealed that the integrin 3, 4, and 5 subunits are also related to vessel formation. Recently, it was reported that some angiogenesis inhibitors associate with integrin subunits. Endostatin associated with integrin 5 and v subunits, and angiostatin, tamustatin, and arresten, carboxy peptides of collagen IV, associated with integrin vß3 and 1ß1 (44
, 45)
. These peptides showed significant antitumor effects in mouse models, although their mechanism of action is unclear. In regard to the regulation of mitogen-activated protein kinase by growth factors, various integrin complexes are thought to function in an anchorage-dependent manner (46)
. Because E7820 decreased the expression level of several integrin subunits such as 2, 3, 5, 6, and ß1 in subconfluent cells, it should be elucidated whether these changes are related to inhibition of proliferation of HUVEC. Recent findings strongly suggest that several integrin subunits and physiological inhibitors regulate the angiogenesis process. A study of the action mechanisms of the above inhibitors and E7820 should greatly improve our understanding of the role of integrins in angiogenesis.
E7820 showed significant efficacy against human colorectal cancer (WiDr cell line) -induced angiogenesis in the DAS model on oral administration and delayed in vivo growth of s.c. implanted WiDr cells in nude mice. We reported previously that angiogenesis induced by WiDr cells was dependent on VEGF (31)
. E7820 also significantly inhibited the growth of LoVo cells, an effect which was strongly suppressed by antibody against VEGF (47)
, suggesting that E7820 is effective against VEGF-dependent angiogenesis and tumor growth. Moreover, there was no effect on body weight change during E7820 treatment, and it was possible to continue administration at the maximal tolerated dose for 6 weeks (data not shown). Furthermore, oral administration of E7820 decreased the level of integrin 2 on platelets in mice, although the number of platelets showed no change. Study of integrin 2 level on tumor-induced neovasculature is also of interest for understanding action mechanism of E7820 and is under investigation. The effect of E7820 on integrin 2 in platelets might be useful as a marker of biological effects and could be helpful to determine an effective dose to achieve long-term survival of cancer patients.
Because angiogenesis involves multiple systems in vivo, the possibility that tolerance to an angiogenesis inhibitor might develop must be considered. Therefore, novel types of angiogenesis inhibitors might be useful to decrease the risk of resistance developing to other angiogenesis inhibitors. Because E7820 has a novel action mechanism, suppression of integrin 2 expression, it might be useful in combination therapy with antagonists against VEGF, which up-regulates integrin 2 (32)
, for example. Antiangiogenic therapy based on regulation of integrin also might be more effective if combinations of integrin inhibitors were used according to the expression patterns of integrin on endothelium. Thus, the discovery of E7820 may provide the basis for a new strategy for antiangiogenic therapy, and clinical evaluation of E7820 seems warranted.
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FOOTNOTES |
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1 These authors contributed equally to this study. 
2 To whom requests for reprints should be addressed, at Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki, Japan. Phone: 81-298-47-5740; Fax: 81-298-47-2037; E-mail: t-wakabayashi{at}hhc.eisai.co.jp
3 The abbreviations used are: VEGF, vascular endothelial growth factor; HUVEC, human umbilical vein endothelial cells; FGF, fibroblast growth factor; bFGF, basic fibroblast growth factor; ECM, extracellular matrix; ES, embryonic stem; PMA, phorbol 12-myristate 13-acetate; FBS, fetal bovine serum; TF, tube formation; SFM, serum-free medium; DAS, dorsal air sac.
Received 4/23/02. Accepted 8/29/02.
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vß3 for angiogenesis. Science (Wash. DC), 264: 569-571, 1994.(v)ß(3) in endothelial cells. J. Biol. Chem., 276: 39562-39568, 2001.2ß1 integrin on MCF-7 breast cancer cells. Exp. Cell Res., 248: 260-271, 1999.[Medline]
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Vß3 during angiogenesis. J. Clin. Investig., 98: 426-433, 1996.[Medline]
chains. J. Biol. Chem., 274: 31223-31228, 1999.This article has been cited by other articles:
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) or confluent culture (
). b and c, alteration of expression levels of integrin subunits on HUVEC with E7820 treatment for 48 h at 50 ng/ml in subconfluent and confluent culture, respectively. Expression level is presented as the relative value to nontreatment with E7820. Data are expressed as means of three to five experiments; bars, ± SD. *, P < 0.05; **, P < 0.01.
): 0, (
): 0.06, (
):0.2, (
):0.6, (
): 2.0 nM PMA. d, integrin 
