[Cancer Research 60, 2973-2980, June 1, 2000]
© 2000 American Association for Cancer Research
Experimental Therapeutics |
Porphyrin Analogues as Novel Antagonists of Fibroblast Growth Factor and Vascular Endothelial Growth Factor Receptor Binding That Inhibit Endothelial Cell Proliferation, Tumor Progression, and Metastasis
David Aviezer1,
Sara Cotton,
Magda David,
Amit Segev,
Nona Khaselev,
Nitsa Galili,
Zeev Gross and
Avner Yayon2
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100 [D. A., S. C., M. D., A. S., A. Y.], and Department of Chemistry, Technion-Israel Institute of Technology, Haifa 32000 [N. K., N. G., Z. G.], Israel
 |
ABSTRACT
|
|---|
Fibroblast growth factors (FGFs) and vascular endothelial growth factor
(VEGF) play a pivotal role in the multistep pathway of tumor
progression, metastasis, and angiogenesis. We have identified a
porphyrin analogue,
5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetra-p-tosylate
salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor binding
and activation. TMPP demonstrated potent inhibition of binding of
soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at
submicromolar concentrations. TMPP inhibits binding of radiolabeled
FGF2 to FGFR in a cell-free system as well as to cells genetically
engineered to express FGFR1. Furthermore, TMPP also inhibits the
binding of VEGF to its tyrosine kinase receptor in a dose-dependent
manner. In an in vitro angiogenic assay measuring the
extent of endothelial cell growth, tube formation, and sprouting, TMPP
dramatically reduced the extent of the FGF2-induced endothelial cell
outgrowth and differentiation. In a Lewis lung carcinoma model, mice
receiving TMPP showed a marked inhibition of both primary tumor
progression and lung metastases development, with nearly total
inhibition of the metastatic phenotype upon alternate daily injections
of TMPP at 25 µg/g of body mass. Finally, novel
meso-pyridylium-substituted, nonsymmetric porphyrins, as
well as a novel corrole-based derivative, with >50-fold increase in
activity in vitro, had a significantly improved efficacy
in blocking tumor progression and metastasis in vivo.
|
INTRODUCTION
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FGFs3
and VEGF act in concert to enhance tumor angiogenesis and metastasis
(1)
. In adults, FGF2 (basic FGF) is highly abundant in
tumors, whereas the oncogenes FGF-4
(hst/kfgf) and FGF-3 (int-2) are found
in tumors such as stomach cancer, Kaposi sarcoma, melanoma, and breast
cancer (2)
. FGFR genes are also frequently
amplified and overexpressed in a variety of cancers such as breast
cancer, gastric adenocarcinomas, melanoma, pancreatic cancers, and many
others (3, 4, 5, 6)
. Recently, activating mutations in FGFRs
were also found to be frequently expressed in both cervical and bladder
carcinomas (7)
.
A most potent and selective angiogenic factor is VEGF. VEGF is a
multifunctional cytokine that plays a key role in physiological and
pathological angiogenesis in vivo by stimulating endothelial
cell proliferation and vessel hyperpermeability (8)
. VEGF
binds to Flt-1 and Flk-1/KDR cell membrane receptors that, similar to
FGFRs, are members of the tyrosine kinase receptor superfamily
(9)
. The association between the growth factor ligands and
their respective receptors stimulates tyrosine kinase activity as one
of the initial biochemical events leading to DNA synthesis and cell
division. Therefore, compounds that inhibit ligand-mediated signal
transduction pathways may be useful for the treatment of cellular
proliferative disorders.
FGF2 and VEGF have synergistic activities on endothelial cell
activation and differentiation (10)
. Both FGF2 and VEGF
require a cooperative interaction with heparan sulfate proteoglycans to
form functional growth factor-receptor complexes that are essential for
high-affinity binding and activation of their cognate receptors
(11, 12, 13)
and angiogenic activity (14)
. On the
basis of the crucial role of growth factor-heparin interaction, we
designed a high throughput screening system using FGF ligands
immobilized on heparin and tagged soluble receptors to identify
molecules that can modulate heparin-FGFR interactions. The screening
process has identified several potent compounds, among which is TMPP, a
member of the porphyrin molecule family.
Porphyrins have been of interest to chemists and medical
scientists for over a century. It has been known for many years that
porphyrins interact with neoplastic tumors (15)
, and the
fact that porphyrins demonstrate high affinity to tumorigenic cells
in vitro and solid tumors in vivo is well
established (16
, 17)
. Moreover, porphyrin
derivatives have been used for the treatment of tumors and malignant
tissues in combination with electromagnetic radiation or radioactive
emissions. Because they strongly absorb light, many porphyrins are
still being used as photosensitizers in photodynamic therapy
(17)
.
In this study, we demonstrate that TMPP
(Mr 1363) is capable of directly
inhibiting FGF2 and VEGF receptor binding. TMPP inhibits endothelial
cell proliferation and differentiation in an in vitro
angiogenesis model and dramatically reduces primary tumor growth and
metastatic spread of Lewis lung carcinoma tumors in mice. A series of
novel, rationally designed porphyrin analogues demonstrated
significantly improved potency in inhibiting FGFR binding in
vitro and improved efficacy in blocking tumor progression and
metastasis in vivo.
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MATERIALS AND METHODS
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Materials
Human recombinant FGF2 and EGF were purchased from R&D systems.
Human recombinant VEGF was generated by H. Weich (GBF,
Braunscwig, Germany). Heparin-coated plates were prepared by Carmeda
(Sweden). FRAP was prepared as described (18)
. EGF
receptor immunoglobulin fusion protein was generated by Y. Yarden as
described (19)
. CHO cells expressing FGFR1 were generated
as described (11)
. DMEM and F12 growth mediums, bovine
calf serum, and glutamine were made by Biological Industries (Bet
Haemek, Israel).
|
Compound Synthesis
|
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Preparation of TMPP
A mixture of 5.7 ml (60 mmol, 4 equivalents) of
4-pyridinecarboxaldehyde and 4.15 ml (60 mol, 4 equivalents) pyrrole
was dissolved in 300 ml of propionic acid, and the mixture was heated
to reflux for 2 h. After cooling to room temperature, the solvent
was evaporated to dryness, and the oily residue was washed by hot
water, neutralized by aqueous ammonia (25%), and washed again with hot
water. The purple solids obtained by this procedure were filtered and
dried. The dry solid material was treated with three portions of 50 ml
of dichloromethane, each followed by filtration. To the combined
organic phases, 10 g of basic alumina (activity II) were added,
and the solvent was evaporated to dryness. Separation of
5,10,15,20-tetra(4-pyridyl)porphyrin was achieved by column
chromatography. Rf (2% ethanol in
CH2Cl2) = 0.18; 1H NMR (CDCl3, 
):
9.04 (d, J = 5.5 Hz, 8H), 8.85 (s, 8H), 8.14
(d, J = 5.5 Hz, 8H), -2.95 (s, 2H). Two
hundred mg (0.3 mmol) of 5,10,15,20-tetra(4-pyridyl)porphyrin were
dissolved in 30 ml of dry DMF, and 9 ml of CH3I
were added in one portion. The reaction was stirred at room temperature
for 10 h, after which the reaction mixture was evaporated to
dryness by high vacuum at room temperature. The resulting crystals are
recrystallized from mixtures of methanol and EtOAc.
|
Preparation of Compound P1012
|
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The noncharged intermediate, 5,10,15,20-tetrakis
(3-pyridyl)porphyrin (chemical abstract registry no. 40882-83-5), was
alkylated with methyl toluene sulfonate. 1H NMR
(CDCl3, ): 9.45 (s, 4H), 9.06 (d,
J = 5.5 Hz, 4H), 8.85 (s, 8H), 8.52 (d,
J = 7.8 Hz, 4H), 7.7 (dd,
J = 7.8 Hz, 4H), -2.86 (s, 2H).
|
Preparation of Compound P1016 and Related Porphyrins
|
|---|
(a) Preparation of the Intermediate Compounds
(Condensation Step).
A mixture of 4.3 ml (45 mmol, 3 equivalents) of
4-pyridinecarboxaldehyde, 2.06 ml (16.5 mmol, 1 equivalent) of
pentafluorobenzaldehyde, and 4.15 ml (60 mmol, 4 equivalents) of
pyrrole was dissolved in 300 ml of acetic acid, and the mixture was
heated to reflux for 2 h. After cooling to room temperature, the
solvent was evaporated to dryness by vacuum, and the oily residue was
washed by hot water, neutralized by aqueous ammonia (25%), and washed
again with hot water. The purple solids obtained by this procedure were
filtered and dried. The dry solid material was treated with three
portions of 50 ml of dichloromethane, each followed by filtration. To
the combined organic phases, 10 g of silica were added, and the
solvent was evaporated to dryness.
|
(b) Chromatographic Separation.
|
|---|
Separation and purification of the components obtained in step
a was achieved by column chromatography, in which the
polarity of the eluents was gradually increased from dichloromethane to
mixtures of dichloromethane and 2–10% ethanol. The order of elution
(the Rf values are for silica with 2%
ethanol in CH2Cl2) and the
chemical yields were as follows:
5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin
(1a = P2, traces;
Rf = 0.95);
5,10,15-tris(2,3,4,5,6-pentafluorophenyl)-20-(4-pyridyl)porphyrin
(1b, 1.1%; Rf = 0.66);
5,15-bis(2,3,4,5,6-pentafluorophenyl)-10,20-bis(4-pyridyl)porphyrin
(1c; Rf = 0.60);
5,10-bis(2,3,4,5,6-pentafluorophenyl)-15,20-bis(4-pyridyl) porphyrin
(1d; Rf = 0.54);
5-(2,3,4,5,6-pentafluorophenyl)-10,15,20-tris(4-pyridyl)porphyrin
(1e, 9.4%; Rf = 0.45); and 5,10,15,20-tetrakis(4-pyridyl)porphyrin (1f,
traces; Rf = 0.18). The
combined yield of compounds 1c and 1d was 13.4%.
Their separation required an additional column in which the eluent was
2% ethanol in dichloromethane. Spectroscopic characteristics of the
compounds (1a and 1f are known compounds):
1a, UV-vis
(CH2Cl2)
max, nm: 412, 506, 586;
1H NMR (CDCl3, ): 8.91
(s, 8H), -2.93 (s, 2H); 19F NMR
(CDCl3, ): -136.9 (dd,
J1 = 22.8 Hz, J2 = 7.0 Hz, 8F), -151.6 (t, J = 20.7 Hz, 4F),
-161.7 (dd, J1 = 22.4 Hz,
J2 = 5.8 Hz, 8F); 1b, UV-vis
(CH2Cl2)
max, nm: 414, 506, 582;
1H NMR (CDCl3, ): 9.06
(d, J = 4.3 Hz, 2H), 8.89 (s, 6H), 8.16 (d,
J = 4.2 Hz, 2H), 8.15 (s, 2H), -2.92 (s,
2H); 19F NMR (CDCl3, ):
137.0 (m, 6F), -151.8 (m (2 overlaying t), 3F), -161.8 (m, 6F);
MS+ m/z: 886.1
(MH+, 100%), MS-
m/z: 884.6 (M-, 40%), ([M - H]-, 60%); 1c, UV-vis
(CH2Cl2)
max, nm: 412, 508, 584;
1H NMR (CDCl3, ): 9.06
(d, J = 4.4 Hz, 4H), 8.89 (s, 4H), 8.85 (s,
4H), 8.15 (d, J1 = 4.5 Hz, 4H), -2.94 (s,
2H); 19F NMR (CDCl3, ):
-137.2 (dd, J1 = 23.2 Hz,
J2 = 7.2 Hz, 4F), -152.0 (t,
J = 20.9 Hz, 2F), -161.9
(J1 = 22.8 Hz, J2 = 7.3 Hz, 4F); MS+ m/z: 797.4
(MH+, 100%), MS-
m/z: 794.9 ([M - H]-, 100%); 1d, UV-vis
(CH2Cl2)
max, nm: 414, 508, 582;
1H NMR (CDCl3, ): 9.06
(d, J = 5.8 Hz, 4H), 8.89 (d,
J = 6.6 Hz, 4H), 8.84 (m, 4H), 8.15 (dd,
J1 = 4.3 Hz, J2 = 1.5 Hz, 4H), -2.90 (s, 2H); 19F NMR
(CDCl3, ): -137.1 (dd,
J1 = 23.4 Hz, J2 = 8.1 Hz, 4F), -152.0 (t, J = 21.1 Hz, 2F),
-161.9 (td, J1 = 22.8 Hz,
J2 = 7.9 Hz, 4F); MS+
m/z: 797.4 (MH+, 100%),
MS- m/z: 794.9 ([M - H]-, 100%); 1e, UV-vis
(CH2Cl2)
max, nm: 416, 510, 586;
1H NMR (CDCl3, ): 9.05
(d, J = 5.4 Hz, 6H), 8.90 (d,
J = 4.8 Hz, 2H), 8.84 (m (unresolved
doublets), 6H), 8.14 (m (unresolved doublets), 6H), -2.92 (s, 2H);
19F NMR (CDCl3, ):
-137.3 (dd, J1 = 22.8 Hz,
J2 = 7.9 Hz, 2F), -152.1 (t,
J = 21.7 Hz, 1F), -162.0
(J1 = 23.0 Hz, J2 = 7.7 Hz, 2F); MS+ m/z: 708.1
(MH+, 100%), MS-
m/z: 706.1 ([M - H]-, 100%); 1f,
1H NMR (CDCl3, ): 9.04
(d, J = 5.5 Hz, 8H), 8.85 (s, 8H), 8.14 (d,
J = 5.5 Hz, 8H), -2.95 (s, 2H).
|
(c) Alkylation Step.
|
|---|
Seventy mg (0.1 mmol) of
5-(2,3,4,5,6-pentafluorophenyl)-10,15,20-tris(4-pyridyl) porphyrin
(compound 1e) or any of the other derivatives
1b–1f were stirred at room temperature with 3 ml (48 mmol)
CH3I in 10 ml of DMF for 12 h, after which
the reaction mixture was evaporated to dryness by high vacuum at room
temperature. The resulting crystals were recrystallized from mixtures
of methanol and EtOAc, thus obtaining: 2d,
5,10-bis(2,3,4,5,6-pentafluorophenyl)-15,20-bis(N-methyl-4-pyridylium)
porphyrin diiodide, UV-vis (H2O)
max, nm: 416, 514, 582; 2e
(compound P1016), UV-vis (H2O)
max, nm: 420, 516, 584;
1H NMR (DMSO-d6,
): 9.48 (d, J = 6.5 Hz, 6H), 9.44 (d,
J = 5.4 Hz, 2H), 9.17 (m, 6H), 9.02 (d,
J = 6.5 Hz, 4H), 8.99 (d,
J = 6.5 Hz, 2H), 4.71 (s, 9 H), -3.13 (s,
2H); 19F NMR
(DMSO-d6, ):
-139.3 (dd, J1 = 24.5 Hz,
J2 = 5.8 Hz, 2H), -153.4 (t,
J = 22.2 Hz, 1H), -162.2
(J1 = 23.0 Hz, J2 = 5.1 Hz, 2H).
|
Preparation of Compound P1020
|
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(a) Preparation of the Precursor,
5,10,15,20-Tetra[(4-(2-pyridyl)-2,3,5,6-tetrafluorophenyl)]porphyrin.
One ml of an 1.6 M n-BuLi solution (1.6 mmol)
was added to a stirred solution of 0.14 ml (1.5 mol) 2-bromopyridine in
8 ml of dry THF under an argon atmosphere at -78°C at such a rate
that the temperature of the reaction mixture did not exceed -70°C.
After the addition was complete, the reaction mixture was stirred for
1 h at -78°C, resulting in a clear yellow solution. Next, a
solution of 0.1 g (0.1 mmol) of
5,10,15,20-tetra(2,3,4,5,6-pentafluorophenyl)porphyrin (1a)
in 5 ml of dry THF was added dropwise. The mixture was stirred for
3 h at -78°C and then hydrolyzed with saturated aqueous
bicarbonate solution. The layers were separated, the aqueous layer was
washed with ether, and the combined ether extracts were dried and
evaporated to a solid residue. The product was purified by column
chromatography on silica gel (2:1 EtOAc:hexane) and recrystallized from
EtOAc:ethanol to give 26–30 mg (20–25% yield) of the pure product as
violet solids. 1H NMR
(CDCl3, ): 9.06 (s, 8H), 8.97 (d,
J = 3.9 Hz, 4H), 8.03 (t,
J = 7.7 Hz, 4H), 7.89 (d,
J = 7.5 Hz, 4H), 7.54 (t,
J = 6 Hz, 4H), -2.82 (s, 2H).
19F NMR (CDCl3, ):
-137.57 (q, J = 24.8 Hz, 8F), 144.11 (q,
J = 24.6 Hz, 8F). MS+
m/z: 1211.4 (MH+, 100%),
MS- m/z: 1208.3 ([M - H]-, 100%).
|
(b) Alkylation of the Precursor to Obtain Compound
P1020.
|
|---|
A mixture of 40 mg (33 mmol) of
5,10,15,20-tetra(4-(2-pyridyl)-2,3,5,6-tetrafluorophenyl)porphyrin and
2.5 ml (40 mmol) of CH3I in 6 ml of freshly
distilled DMF was heated to 70°C for 5 h. After evaporation of
the solvent, the product recrystallized from methanol:ether to give 55
mg (95% yield) of the title compound as violet solids. UV-vis
(H2O) max, nm
(ex103
): 410 (238), 508 (17.6), 574.
1H NMR (DMSO-d6,
): 9.70 (s, 4H), 9.59 (s, 4H), 9.52 (d, J = 5.8 Hz, 4H), 9.04 (t, J = 7.4 Hz, 4H),
8.79 (d, J = 7.6 Hz, 4H), 8.54 (t,
J = 6.8 Hz, 4H), 4.72 (s, 12H), -3.05 (s,
2H). 19F NMR
(DMSO-d6, ): -137.23 (m, 8F),
137.68 (m, 8F).
|
Preparation of Corrole P1021
|
|---|
The preparation of P1021 and its precursors are described by
Gross et al. (20)
. In brief, this is the
preparation of intermediate
5,10,15-tris(4-(2-pyridyl)-tetrafluorophenyl) corrole. 0.42 ml of a 1.6
M n-BuLi solution (0.7 mmol) was added
to a stirred solution of 0.054 ml (0.56 mmol) 2-bromopyridine in 6 ml
of dry THF under an argon atmosphere at -78°C at such a rate that
the temperature of the mixture did not exceed 70°C. After the
addition was complete, the reaction mixture was stirred for 1 h at
78°C to give a clear yellow solution. Next, a solution of 0.03 g
(0.038 mmol) of 5,10,15-tri(2,3,4,5,6-pentafluorophenyl)corrole in 6 ml
of dry THF was added dropwise. The mixture was stirred for 1 h at
-78°C and then hydrolyzed with saturated aqueous bicarbonate
solution. The layers were separated, the aqueous layer was washed with
ether, and the combined ether extracts were dried and evaporated to a
solid residue. The product was purified by column chromatography on
silica gel (1:1 EtOAc:hexane) and recrystallized from
CH2Cl2:hexane to give 13 mg
(35% yield) of the pure product as violet crystals.
|
Binding of Soluble FGFR Alkaline Phosphatase Fusion Protein to
Immobilized FGF2
|
|---|
FGF2 (100 ng/ml) was incubated on 96-well plates to which
heparin had been covalently attached (Carmeda). Subsequently, 200 µl
of FRAP condition medium and TMPP were added and incubated together for
2 h. After three cycles of washing with HNTG [20 mM
HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, and 10%
glycerol], alkaline phosphatase substrate (Sigma; 15 mM)
was added, and catalyzation of the chromogenic product was measured by
spectrophotometry at 405 nm.
|
Binding of 125I-Labeled FGF2 to Soluble FGFR
|
|---|
Conditioned medium from NIH 3T3 cells secreting FGFR1-FRAP was
incubated for 45 min at room temperature with rabbit antihuman
placental AP antibodies prebound to agarose-protein A beads (Pierce).
The FRAP-coupled beads were washed three times with 1 ml of HNTG and
incubated with 2 ng/ml of 125I-labeled FGF, 1
µg/ml heparin, and TMPP at different concentrations for 1 h at
room temperature. High affinity-bound
125I-labeled FGF2 was determined by counting the
tubes in a gamma counter.
|
Binding of 125I-Labeled FGF2 to Cells
|
|---|
Confluent cultures of CHO cells expressing FGFR1
(11)
in 24-well plates (Falcon) were precooled and washed
twice with cold DMEM supplemented with 20 mM HEPES (pH 7.5)
and 0.1% BSA (DMEM/BSA). They were then incubated for 1.5 h at
4°C with 125I-labeled FGF2 (2 ng/ml) and
increasing concentrations of TMPP. The binding medium was discarded,
and the cells were washed once with ice-cold DMEM/BSA and twice with
cold PBS (pH 7.5) containing 1.6 M NaCl. High-affinity,
receptor-bound FGF2 was determined by extraction of the cells with 20
mM sodium acetate (pH 4.0) containing 2.0 M
NaCl. Nonspecific binding was determined in the presence of a 100-fold
excess of unlabeled FGF2.
|
Binding of 125I-Labeled VEGF to Cells
|
|---|
Confluent cultures of bovine aortic endothelial cells in 24-well
plates (Falcon) were precooled and washed twice with cold DMEM
supplemented with 20 mM HEPES (pH 7.5) and 0.1% BSA
(DMEM/BSA). The cells were then incubated for 1.5 h at 4°C with
125I-labeled VEGF (2 ng/ml) and increasing
concentrations of TMPP. The binding medium was discarded, and the cells
were washed once with ice-cold DMEM/BSA and twice with cold PBS (pH
7.5) containing 1.6 M NaCl. High-affinity, receptor-bound
FGF2 was determined by extraction of the cells with 20 mM
sodium acetate (pH 4.0) containing 2.0 M NaCl. Nonspecific
binding was determined in the presence of a 100-fold excess of
unlabeled VEGF.
|
Binding of 125I-Labeled EGF to EGF Receptor
|
|---|
Confluent cultures of A431 cells in 24-well plates (Falcon) were
precooled and washed twice with cold DMEM supplemented with 20
mM HEPES (pH 7.5) and 0.1% BSA (DMEM/BSA). The cells were
then incubated for 1.5 h at 4°C with
125I-labeled EGF (2 ng/ml) and increasing
concentrations of TMPP (19)
. Binding of radiolabeled EGF
to soluble receptors was performed by incubating conditioned medium
from 293 cells secreting EGF receptor-Fc immunoglobulin fusion protein
for 45 min at room temperature with agarose-protein A beads (Pierce).
The EGF receptor-coupled beads were washed three times with 1 ml of
HNTG and incubated with 2 ng/ml of 125I-labeled
EGF and TMPP at for 1 h at room temperature as described by Tzahar
et al. (19)
. High affinity-bound,
125I-labeled EGF was determined by counting the
tubes in a gamma counter.
|
Rat Aorta in Vitro Angiogenesis Assay
|
|---|
Type I collagen was prepared from the tail tendons of adult
Sprague Dawley rats (21)
. The collagen matrix gel was
obtained by simultaneously raising the pH and ionic strength of the
collagen solution (22)
. Thoracic aortas were obtained from
2-month-old Sprague Dawley rats. The fibroadipose tissue was carefully
removed under a dissecting microscope, and aortic rings were sectioned
(1-mm long) and placed on top of a 0.2-ml collagen gel in 16-mm culture
wells. Collagen solution (0.4 ml) was carefully poured on top of the
ring. After the gel was formed, 0.4 ml of serum-free endothelial growth
medium was added and replaced every other day by fresh medium
containing FGF2 (2 ng/ml). TMPP was added to the growth medium twice a
week. Microvessel outgrowth was visualized by phase microscopy, and the
number of capillary vessels was counted throughout the course of the
experiment. After 14 days, the cultures were fixed with 4%
formaldehyde, embedded in paraffin, and sectioned at 5 µm, and the
extent of microvascular endothelial tube outgrowth was measured under a
light microscope.
|
Lewis Lung Carcinoma Tumor Assay
|
|---|
Murine Lewis lung carcinoma D122 cells (2 x 105 cells/50 ml PBS) were injected into the foot
pads of 10-week-old C57 black mice (23)
. Twenty-five
µg/g body mass of porphyrins dissolved in PBS were injected i.p.
twice a week in the treated group, and tumor size was measured
periodically to follow primary tumor formation. To evaluate inhibition
of lung metastasis by TMPP, the primary tumors were allowed to develop
over a period of 4 weeks to a volume of 
8 mm3
,
after which the tumors were removed by amputation, and metastases were
allowed to develop for an additional 4 weeks, during which the treated
group received 25 µg/g body mass of porphyrin i.p. twice a week.
Subsequently, the mice were sacrificed and dissected, and the lungs
were removed and photographed. The extent of lung metastasis was
measured by weighing the lungs. C57/black mice were maintained on lab
chow and tap water and were housed with a 12-h day-night cycle.
|
RESULTS
|
|---|
TMPP Inhibits the Binding of FGF2 to the FGF Receptor.
A high throughput screening system composed of a heparin matrix, FGF2,
and a FGFR1 tagged by alkaline phosphatase (FRAP) was designed using
96-well plates to which heparin had been covalently attached. FGF2
binding to the immobilized heparin is then followed by the addition of
FRAP, and the compounds were to be screened for their ability to
modulate heparin-FGF, receptor-heparin, and receptor-FGF interactions.
The end point of the assay measures enzymatically the formation of
FGF-receptor complexes quantitated by the specifically associated
alkaline phosphatase-catalyzed chromogenic product. Thus, a lowered
alkaline phosphatase activity would indicate inhibition at one or more
of the three levels of interactions required for the formation of the
FGF-FGFR-heparin ternary complex. The screen of the chemical synthetic
library has identified several compounds for their capacity to inhibit
soluble FGFR binding. One of the most potent ones was TMPP.
TMPP demonstrated potent inhibition at submicromolar concentrations,
with a distinct dose-dependent inhibition pattern (Fig. 1
A). To evaluate the capacity of TMPP to inhibit FGF2 receptor
binding, we measured the binding of radiolabeled FGF2 to FGFR1 in two
independent experimental systems. In the first experiment, we used a
cell-free system, measuring the binding of radiolabeled FGF2 to a
dimeric soluble FGFR1 fused to alkaline phosphatase (18)
.
The soluble FRAP was immobilized using an anti-alkaline phosphatase
antibody prebound to agarose-protein A beads. As a second experimental
model, we used heparan sulfate-deficient CHO cells, genetically
engineered to express FGFR1 (11)
. TMPP inhibited binding
of 125I-labeled FGF2 to the FGFR in both
experimental systems. Fig. 1B
illustrates that TMPP is
capable of profoundly inhibiting FGF2-FGFR1 binding in the soluble
receptor assay with an IC50 of 1
µM (Fig. 1B
). In the cellular
receptor system, TMPP was capable of inhibiting FGF2 binding with an
IC50 of 2.5 µM (Fig. 1C
). The slightly higher concentrations of TMPP required for
inhibition of cellular FGF2 binding in the cellular assay result most
likely from the inherent reduced affinity of the soluble FGFR to the
FGF ligand compared with that of cell-associated receptors
(18)
.
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|
Fig. 1. Inhibition of FGF2 binding to the FGFR by TMPP.
A, binding of soluble FRAP to FGF2 immobilized on
heparin-coated 96-well plates. FGF was prebound to heparin-coated
plates; conditioned medium from NIH 3T3 cells expressing the FGFR-1 AP
fusion protein was added to the wells and incubated in the presence of
increasing concentrations of TMPP. The alkaline phosphatase
enzymatically catalyzed formation of a chromogenic product was measured
(405 nm) and represents the amount of FRAP present on the plates as a
heparin-FGF-FRAP ternary complex. B, effect of TMPP on
binding of radiolabeled FGF2 to soluble FGFR1. Radiolabeled FGF2 (2
ng/ml) was incubated (90 min at 4°C) with immobilized FGFR1.
Incubations were performed in the presence of 100 ng/ml heparin and
increasing concentrations of TMPP. Nonspecific binding was determined
in the presence of 100-fold excess of unlabeled FGF2 and did not exceed
10% of the total binding. Results represent the means in one of at
least two independent experiments. C, effect of TMPP on
the binding of 125I-labeled FGF2 to CHO cells transfected
with FGFR1. HS-deficient CHO mutant cells expressing FGFR1 were
incubated for 90 min at 4°C in the presence of 1 µg/ml of heparin,
125I-labeled FGF2 (2 ng/ml), and increasing concentrations
of TMPP. The binding medium was discarded, and the cells were washed
with ice-cold DMEM/BSA. To determine the degree of receptor-bound
125I-labeled FGF2, the cells were incubated in cold PBS (pH
4) containing 1.6 M NaCl and 25 mM HEPES. The
cell extracts were counted in a gamma counter. Nonspecific binding was
determined in the presence of 100-fold excess of unlabeled ligand and
did not exceed 20% of the total bound ligand.
|
|
|
Affinity Labeling of Cells by 125I-Labeled FGF2 Is
Inhibited by TMPP.
|
|---|
To determine the specificity of this effect, chemical cross-linking of
125I-labeled FGF2 to cells was carried out in the
absence and presence of increasing concentrations of TMPP. As shown in
Fig. 2
, there is complete inhibition of the formation of a typical
FGF2-receptor complex at TMPP concentrations as low as 5
µM, in agreement with the direct binding data (Fig. 1C
).
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Fig. 2. Covalent cross-linking of 125I-labeled FGF2 to
CHO cells expressing FGFR is inhibited by TMPP. Binding of
125I-labeled FGF2 to confluent monolayers of
FGFR1-expressing cells was performed as described above in the presence
of increasing concentrations of TMPP. After 90 min, disuccinylimidyl
suberate (0.15 mM in PBS) was added. The protein complexes
were separated by electrophoresis on a 7.5% SDS polyacrylamide gel and
analyzed on X-ray film.
|
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|
TMPP Inhibits the Binding of VEGF to the VEGF Receptor.
|
|---|
Because FGF and VEGF share several similar characteristics and may play
synergistic role in tumor angiogenesis (2
, 8
, 10)
, we
examined the capacity of TMPP to inhibit VEGF binding to its receptor.
Fig. 3
A demonstrates that TMPP efficiently inhibits VEGF binding to
human umbilical vein endothelial cells that express the
Flk-1/KDR VEGF receptor and with high potency. TMPP also
inhibits VEGF binding to bovine aortic endothelial cells expressing
VEGF receptors (data not shown) in a manner similar to the
Flk-1/KDR-transfected cells.
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Fig. 3. A, effect of TMPP on the binding of
125I-labeled VEGF to endothelial cells transfected with the
VEGF receptor. Confluent monolayers of endothelial cells expressing the
VEGF receptor (Flk-1/KDR) were incubated with 125I-labeled
VEGF (2 ng/ml) for 90 min at 4°C in the presence of increasing
concentrations of TMPP. The binding medium was discarded, and the cells
were washed with ice-cold DMEM/BSA. To determine the degree of
receptor-bound 125I-labeled VEGF, the cells were incubated
in cold PBS (pH 4) containing 1.6 M NaCl and 25
mM HEPES. The cell extracts were counted in a gamma
counter. Nonspecific binding was determined in the presence of
increasing concentrations of unlabeled ligand and did not exceed 20%
of the total bound ligand. B, TMPP does not inhibit
covalent cross-linking of 125I-labeled EGF to EGF receptor.
Binding of 125I-labeled EGF to soluble EGF receptor-Fc
immunoglobulin fusion protein was performed as described by Tzahar
et al. (19)
. Binding of
125I-labeled FGF2 to confluent monolayers of
FGFR1-expressing cells was performed as described above. Both binding
experiments were performed in the presence or absence of 10 µg/ml
TMPP. After 90 min, chemical cross-linking was performed by the
addition of disuccinylimidyl suberate (0.15 mM in PBS). The
protein complexes were separated by electrophoresis on a 7.5% SDS
polyacrylamide gel and analyzed on X-ray film.
|
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TMPP Does Not Inhibit the Binding of EGF to the EGF Receptor.
|
|---|
When TMPP was tested for its capacity to inhibit the binding of
radiolabeled EGF to the EGF receptor on A431 cells, no inhibition of
binding was noted, even at mM concentrations of TMPP (data
not shown). To unequivocally determine the specificity of this effect,
chemical cross-linking of 125I-labeled FGF2 to
FGFR1-expressing cells or 125I-labeled EGF to EGF
receptor (19)
was carried out in the absence or presence
of 10 µg/ml TMPP. As shown in Fig. 3
B, TMPP as expected
inhibits FGF2 receptor binding and the formation of a FGF-receptor
complex but had no effect on the binding of
125I-labeled EGF to the EGF receptor, in
agreement with the results of the binding experiment on A431 cells.
|
Inhibition of in Vitro Angiogenesis by TMPP.
|
|---|
To establish the biological effect of TMPP, we examined the compound
for its effects on an in vitro angiogenic assay using rat
aorta sections embedded in a collagen type I gel (21
, 22
, 24) . The assay measures the extent of endothelial cell growth
and microvascular tubules sprouting from the vessel tissue embedded in
the gel. Basal tubule formation can be detected, even when no
additional factors were added. The addition of FGF2 (2 ng/ml)
dramatically increased the degree of cell growth and vascularization.
However, the addition of FGF2 together with 10
µM TMPP dramatically reduced the extent of
endothelial cell growth and differentiation. When 100
µM TMPP was added in the presence of FGF2,
complete inhibition of microvascular tubule sprouting was achieved, and
no endothelial cell growth was observed (Fig. 4
).
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Fig. 4. In vitro angiogenic assay using a rat aorta
section embedded in a collagen gel. Sections of rat aorta were
immobilized in a collagen gel. After the addition of FGF2 to the
medium, in the presence or absence of TMPP, the extent of endothelial
cell growth and microvascular tubules sprouting from the vessel tissue
embedded in the gel was measured. Basal tubule formation can be
detected even when no additional factors were added. The results are
expressed as the percentage of microvascular tubules sprouting in
comparison with the control experiment where FGF2 alone was added.
Bars, SE.
|
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|
TMPP Inhibits Primary Tumor Progression in a Lewis Lung Carcinoma
Tumor Model.
|
|---|
Lewis lung carcinoma D122 cells (200,000 cells/mouse) were injected
into the foot pads of 10-week-old C57 black mice according to O’Reilly
et al. (23)
. Mice that received the TMPP (25
µg/g of body mass) by i.p. injections twice a week for 5 weeks showed
a marked inhibition in primary tumor growth in comparison with the
control group (Fig. 5
). The experiment was repeated five times, and the findings were
reproducible and statistically established. These results indicated
that TMPP is not only active in vitro but is capable of
inhibiting tumor growth in vivo as well.
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Fig. 5. Inhibition of primary tumor growth in the Lewis lung
carcinoma murine tumor model by TMPP. Lewis lung carcinoma cells were
injected into the foot pads of 10-week-old C57 black mice. TMPP (25
µg/g of body mass) was injected i.p. twice a week over a period of 7
weeks, during which time the primary tumor volume was monitored.
Bars, SE.
|
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|
TMPP Inhibits Lung Metastasis in the Lewis Lung Carcinoma Model.
|
|---|
Mice were injected with Lewis lung carcinoma cells (2 x 105 cells/mouse) into the foot pad, and primary
tumors were allowed to develop over a period of 3 weeks to a volume
of 8 mm3
. Subsequently, primary tumors were
removed through amputation, and lung metastases were allowed to develop
for 4 weeks before the mice were sacrificed. The extent of lung
metastasis at this point was examined by gross morphological
examination and by determining the gain in lung weight. Aggressive
metastasis formation is noted in the lungs of control mice (those not
treated with TMPP). In mice treated with TMPP (25 mg/kg body mass), the
lungs were significantly less affected (Fig. 6
) and in some cases indifferent from those of the noninjected control
mice weighing 200 mg, similar to the lungs from the noninjected
control mice (Fig. 6
).
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Fig. 6. Effect of TMPP on lung metastasis in the Lewis lung
carcinoma murine tumor model. Lewis lung carcinoma cells were injected
into the foot pads of 10-week-old C57 black mice, and primary tumors
were allowed to develop over a period of 4 weeks to a volume of 8
mm3. Subsequently, the primary tumors were removed by
amputation, and metastases were allowed to develop for an additional 4
weeks before mice were sacrificed. Twenty-five µg/g body mass TMPP
were injected i.p. twice a week during the 4-week period. The mice were
sacrificed and dissected, and the lungs were removed. The average
extent of lung metastasis was measured by weighing the lungs for gain
of mass. Bars, SE.
|
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|
Novel Porphyrin Derivatives Demonstrate Improved in
Vitro and in Vivo Activity.
|
|---|
To elucidate the structural requirements needed to achieve FGF and VEGF
inhibitory activity, we have synthesized and examined a series of
porphyrin analogues. It became clear that only cationic charged
porphyrins, but not neutral or anionic charged derivatives, are active.
On the basis of the structure of TMPP, we have synthesized novel
meso-pyridylium-substituted porphyrins in which the position
of the N-methyl (the positive charge) was varied from the
para position to ortho and meta, as
well as porphyrins with fewer than four 4-pyridylium substituents. In
this series, the most beneficial effect, as judged by the inhibition of
FGF2 binding (Fig. 7
A), was obtained with P1016, a nonsymmetric porphyrin with
three positive charges. The activity of P1016 in vitro was
50 times higher than that of TMPP, as can be seen in Fig. 7A
. Another derivative, P1020, which contains four positive
charges at more remote positions, was also significantly more active
than TMPP (Fig. 7A
). In contrast, P1012 was 10-fold less
active in inhibiting FGF2 binding, with an
IC50 of 10 µM (data not
shown). In the Lewis lung carcinoma tumor model, however, only P1020,
but not P1016, was more active than TMPP and P1012, which demonstrated
only residual capacity to inhibit FGF2 binding and had no effect
whatsoever in vivo (Fig. 7B
).
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Fig. 7. A, inhibition of FGF2 binding to the
FGFR by porphyrin analogues. Effect of porphyrin analogues on the
binding of radiolabeled FGF2 to soluble FGFR1. 125I-Labeled
FGF2 (2 ng/ml) was incubated (90 min at 4°C) with immobilized FGFR1.
Incubations were performed in the presence of 100 ng/ml heparin and
increasing concentrations of porphyrin analogues TMPP, P1016, P1020,
and P1021. Nonspecific binding was determined in the presence of
100-fold excess of unlabeled FGF2 and did not exceed 10% of the total
binding. Results represent the means in one of at least two independent
experiments. B, inhibition of metastasis growth in the
Lewis lung carcinoma tumor model by porphyrin analogues. Lewis lung
carcinoma cells were injected into the foot pads of 10-week-old C57
black mice, and primary tumors were allowed to develop over a period of
4 weeks to a volume of 8 mm3. After the formation of
primary tumors, they were removed by amputation, and metastases were
allowed to develop for 4 weeks before mice were sacrificed. Porphyrin
analogues TMPP, P1012, P1016, P1020, and P1021 were injected i.p. twice
a week. The mice were sacrificed and dissected, and the lungs were
removed. The average extent of lung metastasis was measured by weighing
the lungs. The graph demonstrates the concentration (mg/kg body mass)
of porphyrin analogue required to achieve inhibition of metastasis
growth. Bars, SE.
|
|
Finally, we tested a novel water-soluble corrole analogue of TMPP
(P1021; Mr 1311.3) for its activity
for both in vitro inhibition of FGF2 binding and for the
inhibition of tumor growth. Indeed, this derivative, having three
positive charges as in P1016 but with the same side groups as in P1020,
displayed the best of both P1016 and P1020 characteristics, because it
was 10-fold more active than TMPP in vitro and 5-fold
more potent in the in vivo tumor models, inhibiting lung
metastasis formation in vivo at a concentration of only 5
mg/kg body weight (Fig. 7B
). These results suggest that
rationally modified porphyrin analogues can serve as highly potent
inhibitors of growth factor activity in vitro and in
vivo.
|
DISCUSSION
|
|---|
We have identified TMPP, a member of the porphyrin family, as a
potent inhibitor of FGF2 and VEGF receptor binding in cells and
cell-free systems. TMPP also dramatically reduced the extent of
FGF2-induced endothelial cell growth and differentiation in an in
vitro angiogenesis model and efficiently blocks Lewis lung
carcinoma murine primary tumor growth and lung metastasis. Novel,
rationally designed TMPP porphyrin analogues demonstrate improved
potency in inhibiting receptor binding in vitro and tumor
progression and metastasis in vivo. Taken together, we have
identified TMPP and its analogues as a novel class of potent inhibitors
of FGF2 and VEGF activity in vitro and in vivo.
The exact mechanism by which TMPP and other related porphyrin-like
molecules inhibit growth factor-receptor binding and activation is not
clear. However, preliminary results suggest that TMPP interferes
with the formation of the trimolecular complex of growth
factor-heparin and the tyrosine kinase receptor (11)
, thus
abrogating receptor signaling. It is interesting to note that TMPP does
not inhibit the binding of EGF, which is not a heparin-binding or
heparin-dependent growth factor (25)
, to its high-affinity
tyrosine kinase
receptor,4
suggesting that interfering with heparin binding may play a key role in
the inhibitory effect of TMPP. Both the FGF ligand and the FGFR contain
heparin-binding domains critical for FGFR activation
(26, 27, 28, 29)
, and several heparin mimetics have been described
as potent inhibitors of FGFR binding and activation (24
, 30)
. TMPP, however, does not resemble in its structure any of
the known heparin mimetics. Nevertheless, the requirement for
positively charged groups and their spatial distribution may mimic a
restricted highly sulfated domain in heparin, thus serving as a heparin
mimetic. Several other inhibitors of FGF and VEGF that have been shown
to inhibit angiogenesis were designed to inhibit the intrinsic tyrosine
kinase activity of the FGF and VEGF growth factor receptors (31
, 32)
. This novel class of FGF and VEGF inhibitors, however, most
likely works via a different molecular mechanism involving direct
interference with growth factor receptor interaction, thus inhibiting
their biological responses.
Porphyrin derivatives are widely used for the treatment of tumors
and malignant tissues in combination with electromagnetic radiation or
radioactive emissions. Because they strongly absorb light in the
690–880 nm region, many porphyrins were suggested for use as
photosensitizers in photodynamic therapy (16)
. Some
porphyrin derivatives are used in combination with electromagnetic
radiation or radioactive emissions for inhibiting angiogenesis
(33)
. It has been suggested that the activity of porphyrin
derivatives as antitumor agents in the absence of electromagnetic
radiation or radioactive emission may be based on their ability to
cleave DNA because of their capacity to bind to DNA and because they
must always include an excitable central Fe or Mn metal
atom. Here we find that porphyrin-like compounds that do not contain a
metal atom can directly interfere with growth factor receptor tyrosine
kinase interactions.
The potent antiproliferative effect of TMPP is of potential clinical
application not only in blocking growth factor-mediated tumor
proliferation but also in other processes of pathological proliferation
such as restenosis, accelerated atherosclerosis, and pathological
angiogenesis as in diabetic retinopathy and arthritis. In support is
the observation that TMPP markedly inhibited the outgrowth of
microvessels from aortic rings embedded in a collagen gel. Furthermore,
TMPP and its analogues are potent inhibitors of vascular smooth muscle
cell proliferation in vitro.4
Studies
are under way to elucidate the inhibitory effect of TMPP on
angiogenesis and restenosis in experimental animal models.
The fact that we were able to improve the potency of the TMPP
lead compound for its FGF and VEGF inhibitory activity, by rationally
modifying specific groups on the porphyrin backbone, is of great
importance. On the basis of the TMPP blueprint, we found that only
cationic charged porphyrins, but not neutral or anionic charged
derivatives, were active. When meso-pyridylium-substituted
porphyrins, as well as porphyrins with fewer than four 4-pyridylium
substituents, were synthesized and tested, the most beneficial effect,
as judged by the inhibition of FGF2 binding (Fig. 7A
), was
obtained with P1016, a nonsymmetric porphyrin with three positive
charges. Another derivative, P1020, which contains four positive
charges at more remote positions, was also significantly more active
(Fig. 7A
). The corrole (P1021), which contains three
positive charges as in P1016 but with the same side groups as in P1020,
displayed the best of both P1016 and P1020 characteristics.
Furthermore, P1021 synthesis takes a much more straightforward approach
than P1016 (20)
. The key to developing highly potent and
specific antitumor agents relies on the ability to perform chemical
modifications along the course of the development process. The vast
knowledge accumulated with regard to the biological and chemical
properties of porphyrins is therefore of great advantage for any
potential medicinal chemistry approach. This fact, along with their
capacity to block growth factor-mediated tumor progression and
angiogenesis, makes these porphyrins highly attractive candidates for
the development of anticancer drugs in the future.
|
ACKNOWLEDGMENTS
|
|---|
We thank Herbert Weich for help with the VEGF receptor binding
assay; Eran Bacharach, Hua-Quan Miao, Israel Vlodavsky, and Eli Keshet
for help with the in vitro angiogenesis assay; Eldad Tzahar
and Yosef Yarden for the soluble EGF receptor; Ezra Vadai and Lea
Eisenbach for help with the Lewis lung carcinoma assay; and Andrew
Seddon and Peter Bohlen for helpful discussions.
|
FOOTNOTES
|
|---|
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.
1 Present address: ProChon Biotech, P. O. Box
1482, Rehovot 76114, Israel. 
2 To whom requests for reprints should be
addressed.
3 The abbreviations used are: FGF, fibroblast
growth factor; FGFR, FGF receptor; FRAP, FGFR1 alkaline phosphatase
fusion protein; VEGF, vascular endothelial growth factor; EGF,
epidermal growth factor; TMPP, 5,10,15,20-tetrakis
N-methyl-4-pyridylium)porphyrin tetraiodide;
P1012,
5,10,15,20-tetrakis(N-methyl-3-pyridylium)-21H,23H-porphine
tetra-p-tosylate; P1016, 5,10,15-tris
(4-N-methylpyridylium)-20-(2,3,4,5,6-pentafluorophenyl)
porphyrin triiodide; P1020,
5,10,15,20-tetra-[4-(N-methyl-2-pyridylium
iodide)-2,3,5,6-tetrafluorophenyl)] porphyrin; P1021,
5,10,15-tris[2,3,5,6-tetrafluoro-4-(N-methyl-2-pyridylium]
corrole triiodide; CHO, Chinese hamster ovary; NMR, nuclear magnetic
resonance; s, singlet; d, doublet; dd, doublet of doublets; t, triplet;
m, multiplet; DMF, dimethylformamide; THF,
tetrahydrofuran.
4 A. Segev, D. Aviezer, M. Safran, Z. Gross, and
A. Yayon, manuscript in preparation.
Received 11/ 8/99.
Accepted 3/29/00.
|
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Targeting protein-protein interactions by rational design: mimicry of protein surfaces
J R Soc Interface,
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[Abstract]
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Y. Ueda, T. Yamagishi, K. Samata, H. Ikeya, N. Hirayama, H. Takashima, S. Nakaike, M. Tanaka, and I. Saiki
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[Abstract]
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S. J. Kwon, A. L. de Boer, R. Petri, and C. Schmidt-Dannert
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[Abstract]
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A. Facchiano, K. Russo, A. M. Facchiano, F. De Marchis, F. Facchiano, D. Ribatti, M. S. Aguzzi, and M. C. Capogrossi
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[Abstract]
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A. Segev, D. Aviezer, M. Safran, Z. Gross, and A. Yayon
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[Abstract]
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D. Aviezer, A. P. Seddon, M. J. Wildey, P. Bohlen, and A. Yayon
Development of a High Throughput Screening Assay for Inhibitors of Fibroblast Growth Factor-Receptor-Heparin Interactions
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[Abstract]
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ABSTRACT
INTRODUCTION
