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Vol. 4, 1405-1414, June 1998 Clinical Cancer Research 1405
443 ‘ -‘ hydroxylphenyl)-amino-6,7-dimethoxyquinazoline:
A Novel Quinazoline Derivative with Potent Cytotoxic Activity
against Human Glioblastoma Cells
Rama Krishna Narla, Xing-Ping Liu,
Dorothea E. Myers, and Fatih M. Uckun1
Departments of Experimental Oncology [R. K. N., F. M. U.],
Biotherapy [R. K. N., D. E. M., F. M. U.], and Chemistry [X-P. L.],and Drug Discovery Program [X-P. L., D. E. M., F. M. U.], HughesInstitute, St. Paul, Minnesota 55 1 13
ABSTRACT
The novel quinazoline derivative 4-(3’-bromo-4’-
hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-
P154) exhibited significant cytotoxicity against U373 and
U87 human glioblastoma cell lines, causing apoptotic cell
death at micromolar concentrations. The in vitro antiglio-
blastoma activity of WHI-P154 was amplified >200-fold
and rendered selective by conjugation to recombinant
human epidermal growth factor (EGF). The EGF-P154
conjugate was able to bind to and enter target glioblas-
toma cells within 10-30 mm via receptor (R)-mediated
endocytosis by inducing internalization of the EGF-R
molecules. In vitro treatment with EGF-P154 resulted in
killing of glioblastoma cells at nanomolar concentrations
with an IC50 of 813 ± 139 flM, whereas no cytotoxicity
against EGF-R-negative leukemia cells was observed,
even at concentrations as high as 100 �LM. The in vivo
administration of EGF-P154 resulted in delayed tumor
progression and improved tumor-free survival in a severe
combined immunodeficient mouse glioblastoma xenograft
model. Whereas none of the control mice remained alive
tumor-free beyond 33 days (median tumor-free survival,
19 days) and all control mice had tumors that rapidly
progressed to reach an average size of > 500 mm3 by 58
days, 40% of mice treated for 10 consecutive days with 1
mg/kg/day EGF-P154 remained alive and free of detect-
able tumors for more than 58 days with a median tumor-
free survival of 40 days. The tumors developing in the
remaining 60% of the mice never reached a size >50
mm3. Thus, targeting WHI-P154 to the EGF-R may be
useful in the treatment of glioblastoma multiforme.
Received 2/4/98; revised 4/1/98; accepted 4/3/98.
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 toindicate this fact.
1 To whom requests for reprints should be addressed, at Hughes Insti-tute, 2665 Long Lake Road, Suite 330, St. Paul, MN 551 13. Phone:(612) 697-9228; Fax: (612) 697-1042.
INTRODUCTION
As the most malignant primary central nervous system
tumors, high-grade anaplastic astrocytoma and glioblastoma
multiforme respond poorly to contemporary multimodality
treatment programs using surgical resection, radiation therapy,
and chemotherapy with a median survival of < 1 year after
initial diagnosis (1-4). Consequently, the development of effec-
tive new agents and novel treatment modalities against these
very poor prognosis brain tumors remains a major focal point in
translational oncology research.
In a systematic effort to identify a cytotoxic agent with
potent antitumor activity against glioblastoma cells, we synthe-
sized several dimethoxy-substituted quinazoline derivatives and
examined their in vitro and in vivo effects on human glioblas-
toma cells. Here, we provide experimental evidence that the
novel quinazoline derivative WHI-P1S42 exhibits potent cyto-
toxic activity against human glioblastoma cells. Notably, target-
ing of WHI-P154 to the surface EGF-R further enhanced its
cytotoxic activity, resulting in rapid apoptotic death of glioblas-
toma cells at nanomolar concentrations in vitro and significantly
improved tumor-free survival in an in vivo SCID mouse
glioblastoma xenograft model.
MATERIALS AND METHODS
Synthesis and Analysis of Quinazoline Derivatives.All chemicals were purchased from the Aldrich Chemical Com-
pany (Milwaukee, WI) and were used directly for synthesis.
Anhydrous solvents such as acetonitrile, methanol, ethanol,
ethyl acetate, tetrahydrofuran, chloroform, and methylene chlo-
ride were obtained from Aldrich as Sure Seal bottles under
nitrogen and were transferred to reaction vessels by cannulation.
All reactions were carried out under a nitrogen atmosphere.
2 The abbreviations used are: WHI-P154, 4-(3’-bromo-4’hydroxylphe-
nyl)-amino-6,7-dimethoxyquinazoline; WHI-P79, 4-(3’-bromophenyl)-amino-6,7-dimethoxyquinazoline; WHI-P97, 4-(3’,5’-dibromo-4’-hy-
droxylphenyl)-amino-6,7-dimethoxyquinazoline: WHI-PI 1 1 . 4-(3’-
bromo-4’-methylphenyl)-amino-6,7-dimethoxyquinazoline: WHI-P131.
4-(4’-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; WHI-P132,
4-(2’-hydroxylphenyl)-arnino-6,7-dimethoxyquinazoline; WHI-Pl80,
4-(3’-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; WHI-P 197,
4-(3’-chloro-4’-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline:
WHI-P258, 4-(phenyl)-amino-6,7-dimethoxyquinazoline; SCID, severecombined immunodeficient; EGF, epidermal growth factor; rhEGF, recom-binant human EGF; EGF-R, EGF receptor; NMR, nuclear magnetic reso-nance; 5, d, t, q, m, and br, singlet, doublet, triplet, quartet. multiplet, and
broad, respectively; IR, infrared; GC/MS. gas chromatography/mass spec-
trometiy; m.p., melting point; HPLC, high-performance liquid chromatog-raphy; Sulfo-SANPAH, sulfosuccinimidyl 6-(4’azido-2’-nitrophenylami-no)hexanoate; MU, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyb tetrazoliumbromide; GEN, genistein: TdT, terminal deoxynucleotidyl transferase:VTK, protein tyrosine kinase.
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Table 1 6,7-Dimethoxyquinazoline derivatives
No R Formular mp(’C) MW�
WHI-P79 3-Br C,,H,4BrN,02 246.0-249.0 360
WHI-P97 3-Br, 5-Br, 4-OH C16H13Br2N,O, >300.0 455
WHI-Pi 1 1 3-Br, 4-CH, C17H16BrN,02 225.0-228.0 374
WHI-P131 4-OH C16H,,N,O, 245.0-248.0 297
WHI-P132 2-OH C16H13N,O, 255.0-258.0 297
WHI-P154 3-Br, 4-OH C,,H14BrN,O, 233.0-233.5 376
WHI-P180 3-OH C16H,,N,O, 256.0-258.0 297
WHI-P197 3-Cl, 4-OH C16H14C1N,O, 245.0(dec) 341
WHI-P258 H C16H,,N,02 258.0-260.0 281
Proton and carbon NMR (‘H and ‘3C NMR) spectra were
recorded on a Mercury 2000 Varian spectrometer operating at 300
and 75 MHz, respectively, using an automatic broad-band probe.
Unless otherwise noted, all NMR spectra were recorded in CDC13
at room temperature. ‘H chemical shifts are quoted in parts per
million (� in ppm) downfield from tetramethyl silane, which was
used as an internal standard at 0 ppm and s, d, t, q, and m. Melting
points were determined using a Fisher-Johns melting apparatus and
are uncorrected. UV spectra were recorded using a Beckman model
DU 7400 UVNis spectrometer with a cell path length of 1 cm.
Methanol was used as the solvent for the UV spectra. Fourier
Transform IR spectra were recorded using an FT-Nicolet model
Protege 460 instrument. The JR spectra of the liquid samples were
run as undiluted liquids using KBr discs. The KBr pellet method
was used for all solid samples. The GC/mass spectrum analysis was
conducted using a Hewlett-Packard GC/mass spectrometer model
6890 equipped with a mass ion detector and Chem Station soft-
ware. The temperature of the oven was steadily increased from
70#{176}Cto 250#{176}C,and the carrier gas was helium.
General Procedure for Synthesis of 6,7-Dimethoxy-
quinazoline Derivatives. The 6,7-dimethoxyquinazoline de-
rivatives for this study were prepared by the condensation of
4-chloro-6,7-dimethoxyquinazoline and the substituted anilines
as outlined in Scheme 1:
CH�Dj�J�N + � ‘ cH�R)fl
R a substituent; n = number
Specifically, a mixture of 4-chloro-6,7-dimethoxyquinazoline
(448 mg, 2 mmol) and the substituted aniline (2.5 mmol) in
ethanol (20 ml) was heated to reflux. Heating was continued for
4-24 h, an excess amount of Et3N was added to the solution,
and the solvent was concentrated to give the crude product,
which was recrystallized from dimethylformamide.
The key starting material, 4-chloro-6,7-dimethoxyquinazo-
line, was prepared using published procedures (5, 6) as outlined
in Scheme 2:
0 0
CH30-(�’jf”0H_(1)SOC�� CH30-�)”#{176}�’NH2 �
CH3O.5�.�.A. (2) NH4OH CH30�I��..k..NO NaBH�
( 1 ) (2)
CH:�1ZI(� HC0�H �
(3) (4)
Specifically, 4,5-dimethoxy-2-nitrobenzoic acid (compound 1)was treated with thionyl chloride, which was directly reduced
with ammonia to yield 4,S-dimethoxy-2-nitrobenzamide (com-
pound 2). Compound 2 was reduced with sodium borohydride in
the presence of catalytic amounts of copper sulfate to give
4,5-dimethoxy-2-aminobenzamide (compound 3), which was
directly refluxed with formic acid to yield 6,7-dimethoxyquina-
zoline-4(3H)-one (compound 4). Compound 4 was refluxed
with phosphorus oxytrichloride to give 4-chloro-6,7-dimethoxy-
quinazoline (compound 5) in good yield.
Quinazoline Derivatives and Their Physical Data. Ta-
ble 1 lists the quinazoline derivatives synthesized for the present
study. Selected analytical data for these compounds and their
precursors are as follows:
4,5-Dimethoxy-2-nitrobenzamide (precursor compound 2).
Yield 88.50%, m.p. 197.0-200.0#{176}C. ‘H NMR (DM50-cU: � 7.60
(s, 2H, �‘�2)’ 7.57 (s, 1H, 6-H), 7.12 (s, 1H, 3-H), 3.90, 3.87 (s, s,
6H, -OCH3). UV (methanol) X,,,..,�(#{128}):206.0, 244.0, 338.0 am. JR
(KBr) u,,�: 3454, 2840, 1670, 15 12, 1274, 1227 cm ‘. GC/MS
m/z 226 (M�, 10.0), 178 (98.5), 163 (100.0), 135 (51.0).
6,7-Dimethoxyquinazoline-4(3H)-one (precursor com-
pound 4). Yield 81.50%, m.p. 295.0-297.0#{176}C. ‘H NMR
(DMSO-d6): S 12.03 (br, 5, 1H, -NH), 7.99 (s, 1H, 2-H), 7.42 (s,
1H, S-H), 7.1 1 (s, lH, 8-H), 3.88, 3.85 (s, s, 6H, -OCH3). UV
(methanol) Xmax(� 212.0, 241.0 nm. JR (KBr) vm�: 3015,
2840, 1648, 1504, 1261, 1070 cm’. GC/MS mlz 206 (M�,
100), 191 (M� -CH3, 31.5), 163 (16.7), 120 (15.2).
4-Chloro-6,7-dimethoxyquinazoline (precursor compound
5). Yield 75.00%, m.p. 259.0-263.0#{176}C. ‘H NMR (DMSO-d6): �
8.75 (s, 1H, 2-H), 7.53 (s, 1H, S-H), 7.25 (s, 1H, 8H), 3.91 (s,
3H, -OCH3), 3.89 (s, 3H, -OCH3); UV (methanol) Xmax(�
207.0, 212.0, 241 .0 nm. IR (KBr) Umax: 2963, 2834, 1880, 1612,
1555, 1503, 1339, 1153, 962cm’. GC/MS m/z 224(M�, 100),
209 (M�-CH3, 9.4), 189 (19.39), 169 (10.55). Anal.
(C,QH9C1N202) C, H, N.
4-(3 ‘-Bromophenyl)-amino-6,7-dimethoxyquinazoline
(WHI-P79). Yield 84.17%, m.p. 246.0-249.0#{176}C. ‘H NMR
(DMSO-d6): S 10.42 (br, s, 1H, NH), 8.68 (s, 1H, 2-H), 8.07-
7.36 (m, SH, 5, 2’, 4’, 5’, 6’-H), 7.24 (s, 1H, 8H), 3.98 (s, 3H,
1406 Activity of WHI-Pl54 against Human Glioblastoma Cells
(5)
CH30-r�’j(�N
CH30-�L.�i
CH3O�J�J(’L)N( A),
CH�0 �
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Clinical Cancer Research 1407
was used to generate a UV spectrum for each of the samples to
-OCH3), 3.73 (s, 3H, -OCH3 ); UV (methanol) Xmax(� 217.0,
227.0, 252.0 nm; IR (KBr) Umax 3409, 2836, 1632, 1512, 1443,
1243, l068cm’.GCIMSm/z36l (M� + 1, 61.83), 360 (Mt,
100.00), 359 (M�-l, 63.52), 344 (11.34), 222 (10.87), 140
(13.65). Anal. (C,6H,4BrN3O2) C, H, N.
4-(3 ‘,S ‘-Dibromo-4’-hydroxylphenyl)-amino-6,7-dimeth-
oxyquinazoline (WHI-P97). Yield 72.80%, m.p. >300.0#{176}C. ‘H
NMR (DMSO-d6): � 9.71 (s, lH, -NH), 9.39 (s, lH, -OH), 8.48
(s, lH, 2-H), 8.07 (s, 2H, 2’, 6’-H), 7.76 (s, 1H, 5-H), 7.17 (s,
lH, 8-H), 3.94 (s, 3H, -OCH3), 3.91 (s, 3H, -OCH3). UV
(methanol) �‘max(� 208.0, 210.0, 245.0, 320.0 nm; IR (KBr)
Um� 3504 (br), 3419, 2868, 1627, 1512, 1425, 1250, 1155
cm ‘. GC/MS m/z 456 (M� + 1, 54.40), 455 (M�, 100.00), 454
(M�-l, 78.01), 439 (M�-OH, 7.96), 376 (M�+ 1-Br, 9.76), 375
(Mt-Br, 10.91), 360 (5.23). Anal. (C,6H,3Br2N3O3) C, H, N.
4-(3’-Bromo-4’-methylphenyl)-amino-6,7-dimethoxyquin-
azoline (WHI-Pl 1 1). Yield 82.22%, m.p. 225.0-228#{176}C. ‘H
NMR (DMSO-d6): � 10.23 (s, lH, -NH), 8.62 (s, lH, 2-H), 8.06
(d, lH, f2.,6. = 2.1 Hz, 2’-H), 7.89 (s, 1H, 5-H), 7.71 (dd, lH,
f5..6. = 8.7 Hz, f2.,6. = 2.1 Hz, 6’-H), 7.37 (d, lH, f5.6 8.7
Hz, 5’-H), 7.21 (s, 1H, 8-H), 3.96 (s, 3H, -OCH3), 3.93 (s, 3H,
-OCH3). UV (methanol) �“max(�) 204.0, 228.0, 255.0, 320.0 nm.
IR (KBr) Um� 3431, 3248, 2835, 1633, 1517, 1441, 1281, 115S
cm’. GCIMS m/z 375 (M�+ 1, 76.76), 374 (M�, 100.00), 373
(M�-l, 76.91), 358(M�+ 1-OH, 1 1.15), 357 (1.42), 356 (6.31).
Anal. (C,7H,6BrN3O2.HCI) C, H, N.
4-(4’-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline
(WHI-P13l). Yield 84.29%, m.p. 245.0-248.0#{176}C. ‘H NMR
(DMSO-d6): � 11.21 (s, 1H, -NH), 9.70 (s, 1H, -OH), 8.74 (s,
lH, 2-H), 8.22 (s, 1H, 5-H), 7.40 (d, 2H, J = 8.9 Hz, 2’,6’-H),
7.29(s, 1H, 8-H), 6.85(d, 2H, J 8.9 Hz, 3’, 5’-H), 3.98 (s, 3H,
-OCH3), 3.97 (s, 3H, -OCH3). UV (methanol) Xmax(#{128}):203.0,
222.0, 251.0, 320.0 nm. JR (KBr)Um�: 3428, 2836, 1635, 1516,
1443, 1234 cm ‘ . GC/MS m/z 298 (M� + 1 , 100.00), 297 (M�,
26.56), 296 (Mtl, 12.46). Anal. (C,6H,5N303.HC1) C, H, N.
4-(2’-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline
(WHI-Pl32). Yield 82.49%, m.p. 255.0-258.0#{176}C. ‘H NMR
(DMSO-d6): � 9.78 (s, lH, -NH), 9.29 (s, lH, -OH), 8.33 (s, lH,
2-H), 7.85 (s, 1H, 5-H), 7.41-6.83 (m, 4H, 3’, 4’, 5’, 6’-H), 7.16
(s, 1H, 8-H), 3.93 (s, 3H, -OCH3), 3.92 (s, 3H, -OCH3). UV
(methanol) �“max(� 203.0, 224.0, 245.0, 335.0 nm. IR (KBr)
Um� 3500 (br), 3425, 2833, 1625, 1512, 1456, 1251, 1068
cm’. GC/MS m/z 298 (M�+l, 8.91), 297 (M�, 56.64), 281
(M�+l- OH, 23.47), 280 (Mt- OH, 100.00). Anal.
(C,6H,5N303.HC1) C, H, N.
4-(3 ‘-Bromo-4’-hydroxylphenyl)-amino-6,7-dimethoxy-
quinazoline (WHI-P154). Yield 89.90%, m.p. 233.0-233.5#{176}C.
‘H NMR (DMSO-d6): � 10.08 (s, 1H, -NH), 9.38 (s, lH, -OH),
8.40 (s, lH, 2-H), 7.89 (d, 1H, f2.6 = 2.7 Hz, 2’-H), 7.75 (s,
1H, 5-H), 7.55 (dd, lH, f5.6 = 9.0 Hz, f2.6 = 2.7 Hz, 6’-H),
7.14 (s, 1H, 8-H), 6.97 (d, lH, f5.6 9.0 Hz, 5’-H), 3.92 (s,
3H, -OCH3), 3.90 (s, 3H, -OCH3). UV (methanol) �‘max(�
203.0, 222.0, 250.0, 335.0 nm. IR (KBr) Umax 3431 (br), 2841,
1624, 1498, 1423, l244cm’. GCIMS mlz 378(M�+2, 90.68),
377 (M�+ 1, 37.49), 376 (M�, 100.00), 360 (Mt, 3.63), 298
(18.86), 282 (6.65). Anal. (C,6H14BrN3O3.HC1) C, H, N.
4-(3 ‘-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline
(WHI-P180). Yield 71.55%, m.p. 256.0-258.0#{176}C. ‘H NMR
(DMSO-d6): � 9.41 (s, lH, -NH), 9.36 (s, lH, -OH), 8.46 (s, lH,
2-H), 7.84 (s, lH, 5-H), 7.84-6.50 (m, 4H, 2’, 4’, 5’, 6’-H), 7.20
(s, 1H, 8-H), 3.96 (s, 3H, -OCH3), 3.93 (s, 3H, -OCH3). UV
(methanol) Xmax(� 204.0, 224.0,252.0, 335.0 nm. IR (KBr)
Um�: 3394, 2836, 1626, 1508, 1429, 1251 cm’. GCIMS m/z
297 (M�, 61.89), 296 (Mtl, 100.00), 280 (MtOH, 13.63).
Anal. (C,6H,5N301.HC1) C, H, N.
4-(3 ‘-Chloro-4’-hydroxylphenyl)-amino-6,7-dimethoxy-
quinazoline (WHI-P197). Yield 84.14%, m.p. 245.0#{176}C(dec). ‘H
NMR (DMSO-d6): �l 10.00 (s, 1H, -NH), 9.37 (s, 1H, -OH), 8.41
(s, 1H, 2-H), 7.78 (s, lH, 5-H), 7.49 (d, lH, J2.�,. 2.7 Hz,
2’-H), 7.55 (dd, lH, f56 = 9.0 Hz, f2.6 2.7 Hz, 6’-H), 7.16
(s, lH, 8-H), 6.97 (d, lH, f5.6 9.0 Hz, 5’-H), 3.93 (s, 3H,
-OCH3), 3.91 (s, 3H, -OCH3). UV (methanol) Xmax(#{128}):209.0,
224.0,249.0, 330.0 nm. JR (KBr) Umax: 3448, 2842, 1623, 1506,
1423, 1241 cm’. GC/MS m/z 341 (M�, 100.00), 326 (Mt
CH3, 98.50), 310 (MtOCH3 12.5), 295 (9.0), 189 (13.5), 155
(13.8). Anal. (C,6H14C1N303.HC1) C, H, N.
4-(Phenyl)-amino-6,7-dimethoxyquinazoline (WHI-
P258). Yield 88.26%, m.p. 258.0-260.0#{176}C. ‘H NMR (DM50-
d6): � 11.41 (s, lH, -NH), 8.82 (s, lH, 2-H), 8.32 (s, lH, 5-H),
7.70-7.33 (m, SH, 2’, 3’, 4’, 5’, 6’-H), 7.36 (s, lH, 8H), 4.02
(s,3H, -OCH3), 4.00 (s, 3H, -OCH3). UV (methanol) �‘max(�
210.0, 234.0, 330.0 nm. IR (KBr) Umax: 2852, 1627, 1509, 1434,
1248 cm’. GCIMS m/z 282 (M�+ 1, 10.50), 281 (M�, 55.00),
280 (M�-l, 100.00), 264 (16.00), 207 (8.50). Anal.
(C,6H,5N3O2) C, H, N.
Preparation of the EGF-P154 Conjugate. rhEGF was
produced in Escherichia coli harboring a genetically engineered
plasmid that contains a synthetic gene for human EGF fused at
the NH2 terminus to a hexapeptide leader sequence for optimal
protein expression and folding. rhEGF fusion protein precipi-
tated in the form of inclusion bodies, and the mature protein was
recovered by trypsin cleavage, followed by purification using
ion exchange chromatography and HPLC. rhEGF was 99% pure
by reverse-phase HPLC and SDS-PAGE with an isoelectric
point of 4.6 ± 0.2. The endotoxin level was 0.172 EU/mg. The
recently published photochemical conjugation method using the
hetero-bifunctional photoreactive cross-linking agent, Sulfo-
SANPAH (Pierce Chemical Co., Rockford, IL; Ref. 7), has been
used in the synthesis of the EGF-Pl54 conjugate. Sulfo-
SANPAH-modified rhEGF was mixed with a 10: 1 molar ratio
of WHI-P154 (50 inst solution in DMSO) and then irradiated
with gentle mixing for 10 mm with UV light at wavelengths
254-366 nm with a multiband UV light emitter (model
UVGL-15 Mineralight; UVP, San Gabriel, CA). Photolytic gen-
eration of a reactive singlet nitrene on the other terminus of
EGF-SANPAH in the presence of a 10-fold molar excess of
WHI-P154 resulted in the attachment of WHI-P1S4 to EGF.
Excess WHI-P154 in the reaction mixture was removed by
passage through a prepacked PD-lO column, and l2-kDa EGF-
EGF homoconjugates with or without conjugated WHI-Pl54, as
well as higher molecular weight reaction products, were re-
moved by size-exclusion HPLC. Reverse-phase HPLC using a
Hewlett-Packard 1 100 series HPLC instrument was used for
separation of EGF-P154 from EGF-SANPAH. After the final
purification, analytical HPLC was performed using a Spherisorb
ODS-2 reverse-phase column (250 X 4 mm; Hewlett-Packard).
Prior to the HPLC runs, a Beckman DU 7400 spectrophotometer
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1408 Activity of WHI-P154 against Human Glioblastoma Cells
ascertain the Xmax for EGF-Pl54, EGF-SANPAH, and unmod-
ified EGF. Each HPLC chromatogram was subsequently run at
wavelengths of 214, 265, and 480 nm using the multiple wave-
length detector option supplied with the instrument to ensure
optimal detection of the individual peaks in the chromatogram.
Analysis was achieved using a gradient flow consisting of
0-100% eluent in a time interval of 0-30 mm. Five-pA samples
applied to the above column were run using the following
gradient program: 0-5 mm, 0-20% eluent; 5-20 mm, 20-100%
eluent; 25-30 mm, 100% eluent; and 30-35 mm, 100-0%
eluent. The eluent was a mixture of 80% acetonitrile (CH1CN),
20% H2O, and 0. 1% trifluoroacetic acid. Electrospray ionization
mass spectrometry (8, 9) was performed using a triple quadruple
mass spectrometer (PE SCIEX API; Finnigan, Norwalk, CT) to
determine the stoichiometry of P154 and EGF in EGF-P1S4.
Cell Lines. Human glioblastoma cell lines U87 and U373
were used as targets for various cytotoxic agents, including EGF-
P154. These brain tumor cell lines were obtained from American
Type Culture Collection (Rockville, MD) and maintained as con-
tinuous cell lines in DMEM supplemented with 10% fetal bovine
serum and antibiotics. The B-lineage acute lymphoblastic leukemia
cell line Nalm-6 was used as a negative control.
Uptake and Internalization of EGF-P154. Immunoflu-
orescence was used to: (a) examine the surface expression of
EGF-R on brain tumor cells; (b) evaluate the uptake of EGF-Pl54
by brain tumor cells; and (c) examine the morphological features of
EGF-P154-treated brain tumor cells. For analysis of EGF-R ex-
pression and cellular uptake of EGF-P1S4, U87 and U373 glio-
blastoma cells were plated on poly-L-lysine-coated, glass-bot-
tomed, 35-mm Petri dishes (Mattek Corp., Ashland, MA) and
maintained for 48 h. In uptake studies, the culture medium was
replaced with fresh medium containing S �g/mi EGF, EGF-P154,
or unconjugated WHI-Pl54, and cells were incubated at 37#{176}Cfor
5, 10, 15, 30, and 60 mm and 24 h. At the end of the incubation,
cells were washed twice with PBS and fixed in 2% paraformalde-
hyde. The cells were permeabilized, and nonspecific binding sites
were blocked with 2.5% BSA in PBS containing 0.1% Triton
x- 100 for 30 mm. To detect the EGF-R/EGF-P154 complexes,
cells were incubated with a mixture of a monoclonal antibody (I :10
dilution in PBS containing BSA and Triton X-lOO) directed to the
extracellular domain of the human EGF-R (Santa Cruz Biotech-
nobogies, Inc., Santa Cruz, CA) and a polyclonal rabbit anti-Pl54
antibody (dilution, 1:500) for 1 h at room temperature. After rinsing
with PBS, cells were incubated for 1 h with a mixture of a goat
anti-mouse IgG antibody conjugated to FITC (Amersham Corp.,
Arlington Heights, IL) and donkey anti-rabbit IgG conjugated to
Texas Red (Amersham Corp.) at a dilution of 1:40 in PBS. Simi-
larly, tubulin expression was examined by immunofluorescence
using a monocbonal antibody against a-tubulin (Sigma Chemical
Co., St. Louis, MO) at a dilution of 1:1000 and an anti-mouse IgG
conjugated to FITC. Cells were washed in PBS and counterstained
with TOTO-3 (Molecular Probes, Inc., Eugene, OR) for 10 mm at
a dilution of 1: 1000. Cells were washed again with PBS, and the
coverslips were mounted with Vectashield (Vector Labs, Burl-
ingame, CA) and viewed with a confocal microscope (Bio-Rad
MRC 1024) mounted in a Nikon Labhophot upright microscope.
Digital images were saved on a Jaz disc and processed with Adobe
Photoshop software (Adobe Systems, Mountain View, CA).
Cytotoxicity Assay. The cytotoxicity of various com-
pounds against human brain tumor cell lines was performed using
the MU assay (Boehringer Mannheim Corp., Indianapolis, IN).
Briefly, exponentially growing brain tumor cells were seeded into
a 96-well plate at a density of 2.5 X 10” cells/well and incubated
for 36 h at 37#{176}Cbefore drug exposure. On the day of treatment,
culture medium was carefully aspirated from the wells and replaced
with fresh medium containing the quinazoline compounds WHI-
P79, WHI-P97, WHI-Pl3l, and WFII-Pl54, unconjugated EGF, or
EGF-P154 as well as the tyrosine kinase inhibitory isoflavone
GEN, at concentrations ranging from 0.1 to 250 p.M. Triplicate
wells were used for each treatment. The cells were incubated with
the various compounds for 24-36 h at 37#{176}Cin a humidified 5%
CO, atmosphere. To each well, 10 p.1 of MTF (final concentration,
0.5 mg/mI) was added, and the plates were incubated at 37#{176}Cfor
4 h to allow MIT to form formazan crystals by reacting with
metabolically active cells. The formazan crystals were solubilized
overnight at 37#{176}Cin a solution containing 10% SDS in 0.01 M HC1.
The absorbance of each well was measured in a microplate reader
(Labsystems) at 540 am and a reference wavelength of 690 nm. To
translate the A5�, values into the number of live cells in each well,
the A� values were compared with those on standard A5� versus
cell number curves generated for each cell line. The percentage of
survival was calculated using the formula: % survival - Live cell
number[test]/Live cell number [control] X 100. The IC50s were
calculated by nonlinear regression analysis using Graphpad Prism
software version 2.0 (Graphpad Software, Inc., San Diego, CA).
In Situ Detection of Apoptosis. The demonstration of
apoptosis was performed by the in situ nick-end labeling method
using the ApopTag in situ detection kit (Oncor, Gaithersburg,
MD) according to the manufacturer’ s recommendations. Expo-
nentially growing cells were seeded in six-well tissue culture
plates at a density of SO X l0� cells/well and cultured for 36 h
at 37#{176}Cin a humidified 5% CO2 atmosphere. The supernatant
culture medium was carefully aspirated and replaced with fresh
medium containing unconjugated EGF or EGF-Pl54 at concen-
trations of 10, 25, or SO pg/ml. After a 36-h incubation at 37#{176}C
in a humidified 5% CO2 incubator, the supernatants were care-
fully aspirated, and the cells were treated for 1-2 mm with 0.1%
trypsin. The detached cells were collected into a 15-mb centri-
fuge tube, washed with medium, and pelletted by centrifugation
at 1000 rpm for S mm. Cells were resuspended in 50 p.1 of PBS,
transferred to poly-L-lysine-coated coverslips, and allowed to
attach for 15 mm. The cells were washed once with PBS and
incubated with equilibration buffer for 10 mm at room temper-
ature. After removal of the equilibration buffer, cells were
incubated for 1 h at 37#{176}Cwith the reaction mixture containing
TdT and digoxigenin-l l-UTP for labeling of exposed 3’-hy-
droxyl ends of fragmented nuclear DNA. The cells were washed
with PBS and incubated with antidigoxigenin antibody conju-
gated to FITC for 1 h at room temperature to detect the incor-
porated dUTP. After washing the cells with PBS, the coverslips
were mounted onto slides with Vectashield containing pro-
pidium iodide (Vector Labs, Burlingame, CA) and viewed with
a confocal laser scanning microscope. Nonapoptotic cells do not
incorporate significant amounts of dUTP due to lack of exposed
3 ‘-hydroxyl ends and consequently have much less fluorescence
than apoptotic cells, which have an abundance of exposed
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
150’
125�
-a- DMSO-a-. WHI.P79
WHI.P97
-.- WHI.P131
-0-’ WHI�P154‘-0- GEN
.-�-. WHI�P111
L
-.,- WHI.P197
-0- WH�P258
0
C0
0
0
CC>
C/)
a,0
75,
50�
25
0
10 100 1000 10 100 1000
Clinical Cancer Research 1409
3 x-P. Liu and F. M. Uckun. Inhibitors of EGF-receptor tyrosine kinase,manuscript in preparation.
Fig. 1 Activity of WHI-Pl54 and other
quinazoline compounds on U373 gliobbas-toma cells. Cells were incubated withWHI-P79, WHI-P97, WHI-PI 1 1, WHI-P131, WHI-Pl32, WHI-Pl54, WHI-Pl80,WHI-Pl97, WHI-P258, or GEN for 24 h in96-well plates, and the cell survival wasdetermined by the MTF assay. Data points
represent the means from three independ-ent experiments; bars, SE. The mean (±SE) IC30s were >250 p.M for WHI-P79,
161.2 ± 22.2 p.M for WHI-P97, >250 p.M
for WHI-P131, 167.4 ± 26.9 p.M for WHI-P154, >250 p.M for GEN, >250 p.M forWHI-Pl 1 1, >250 p.M for WHI-P132,178.1 ± 7.4 p.M for WHI-Pl80, >250 p.M
for WHI-P197, and >250 p.M for WHI-
P258. At 250 p.M, the percent survival val-ues were 69.9 ± 5.1% for WHI-Pl3l and43.9 ± 5.5% forWHI-P154 (P 0.02). At
125 p.M, the % survival values were 75.7 ±
5.2% for WHI-P13l and 53.2 ± 5.5% forWHI-P154 (P = 0.04).
3’-hydroxyl ends. In control reactions, the TdT enzyme was
omitted from the reaction mixture.
Maintenance of SCID Mouse Colony. The SCID mice
were housed in a specific pathogen-free room located in a secure
indoor facility with controlled temperature, humidity, and noise
levels. The SCID mice were housed in microisolater cages, which
were autoclaved with rodent chow. Water was also autoclaved and
supplemented with trimethoprim/sulfomethoxazol 3 days/week.
SCID Mouse Xenograft Model of Human Glioblastoma.
The right hind legs of the CB.17 SCID mice were inoculated s.c.
with 0.5 x 106 U373 human glioblastoma cells in 0.2 ml PBS.
SCID mice challenged with brain tumor cells were treated with
EGF-Pl54 (0.5 mg/kg/dose or 1 mg/kg/dose in 0.2 ml of PBS) as
daily i.p. doses for 10 treatment days, starting the day after inocu-
lation of the glioblastoma cells. Daily treatments with PBS, uncon-
jugated EGF (1 mg/kg/dose), and unconjugated W1-ll-Pl54 (1
mg/kg/dose) were used as controls. Mice were monitored daily for
health status and tumor growth and were sacrificed if they became
moribund, developed tumors that impeded their ability to attain
food or water, or at the end of the 3-month observation period.
Tumors were measured using Vernier calipers twice weekly, and
the tumor volumes were calculated according to the following
formula (10): (width2 X length)/2. For histopathological studies,
tissues were fixed in 10% neutral buffered formalin, dehydrated,
and embedded in paraffin by routine methods. Glass slides with
affixed 6-p.m tissue sections were prepared and stained with H&E.
Primary end points of interest were tumor growth and tumor-free
survival outcome. Estimation of life table outcome and compari-
sons of outcome between groups were done, as reported previously
(11-13).
RESULTS AND DISCUSSION
In Vitro Cytotoxicity of WHI-P154 against Human
Glioblastoma Cells. As shown in Fig. 1, WFII-P154, but not the
Drug Concentration (�.tM)
unsubstituted parent quinazoline compound WFH-P258, exhibited
significant cytotoxicity against the U373 human glioblastoma cell
line in three of three independent experiments with a mean (± SE)
IC50 of 167.4 ± 26.9 p.M and a composite survival curve IC50 of
158.5 p.M. The 4’-hydroxyl substituent on the phenyl ring was
essential for the anti-brain tumor activity of WHI-P154 because
WHI-P79, which differs from WHI-Pl54 only by the lack of the
4’-hydroxyl group on the phenyl ring, failed to cause detectable
cytotoxicity to U373 cells (Fig. 1). Replacement of the 4’-hydroxyl
group with a 4’-methyl group (W1-ll-Plll) resulted in substantial
loss of activity (Fig. 1B). The 3’-bromo substitution on the phenyl
ring likely contributes to the cytotoxicity of WHI-Pl54 because
WHI-Pl3l, lacking this bromo substituent, was less potent than
WHI-P154 (IC50s: >250 p�M versus 167.4 ± 26.9 p.M). Similarly,
2’-hydroxylphenyl (WHI-P132) and 3’-hydroxylphenyl (WH1-
P180) derivatives were less active than WHI-P154. Notably, the
replacement of the 3’-bromo substitution with a 3’-chloro substi-
tution yielded a less active agent (WHI-Pl97). Introduction of a
second bromo group at the 5’ position of the phenyl ring did not
result in altered cytotoxicity; the IC50 of WHI-P97 was 161.2 ±
22.2 p.M, which is virtually identical to that of WHI-Pl54 (Fig. 1).
In experiments not shown in this report, WHI-Pl54 was
found to be a potent inhibitor of the EGF-R tyrosine kinase as
well as Src family tyrosine kinases.3 Therefore, we initially
postulated that the cytotoxicity of WHI-Pl54 against glioblas-
toma cells was due to its tyrosine kinase inhibitory properties.
However, to our surprise, WHI-P79, which is an equally potent
inhibitor of EGF-R and Src family tyrosine kinases (14, 15),
failed to cause any detectable cytotoxicity to U373 glioblastoma
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
U373 �
,�..�.. .. , � ,.
.� �
4r., �
A
U87
B
1410 Activity of WHI-P154 against Human Glioblastoma Cells
Fig. 2 Cell surface expression of EGF receptor on U373 and U87human glioblastoma cells. Cells were fixed in paraformaldehyde, im-munostained with monocbonal antibody to EGF-R (green fluorescence),and counterstained with TOTO-3 (blue fluorescence). The immuno-stained cells were analyzed with a laser scanning confocal microscope.Blue fluorescence represents nuclei.
cells. Thus, the cytotoxicity of WHI-Pl54 to U373 cells cannot
be explained by its tyrosine kinase inhibitory properties alone.
This notion was further supported by the inability of the PTK
inhibitor GEN, which was included as a control, to cause de-
tectable cytotoxicity to U373 cells (IC50, >250 p.M; Fig. 1).
Enhanced Cytotoxicity of EGF-conjugated WHI-P154
against Human Glioblastoma Cells. In contrast to normal
glial cells and neurons, a significant portion of glioblastomas
express the EGF-R at high levels (16-20). Therefore, the
EGF-R could be used as a target for delivering cytotoxic agents
to glioblastoma cells with greater efficiency [reviewed by Men-
delsohn and Baselga (21)]. We confirmed the surface expression
of the EGF-R on the U373 and U87 human glioblastoma cell
lines with immunofluorescence and confocal laser scanning
microscopy using monoclonal antibodies to the extracellular
domain of the EGF-R. Both cell lines showed a diffuse granular
immunoreactivity with the anti-EGF-R antibody (Fig. 2).
In an attempt to enhance the antitumor activity of WHI-
P154 against glioblastoma cells by improving its targeting to
and cellular uptake by glioblastoma cells, we conjugated it to
rhEGF. We first examined the kinetics of uptake and cytotox-
icity of the EGF-P154 conjugate in U373 glioblastoma cells
Control ‘�. , � .
.�,. �t
�
‘�.-) � �:
�‘��i: �
A
r�
A’
WH�154: 30 ��
�.�
B ,
�i.
B’
EGF-P154 1Ormn�
�. . , .,.�
‘�*q�.#
‘�r�
C �
. .i�
� , ,
C’
EGF-P154: 30 mm
!�
� .- , ., i.’-4 �
D,,..
D’Fig. 3 EGF-R-mediated uptake of EGF-P154 by U373 human glio-blastoma cells. U373 cells were incubated with unconjugated WHI-
P154 (B and B’) or EGF-P154 (5 p.g/ml; C, C’, D, and D’) for theindicated times and processed for immunofluorescence to detect inter-nalized EGF-RJEGF-P154 complex using a monoclonal antibody to theEGF-R (green fluorescence) and a polyclonal antibody to WI-H-Pl54(red fluorescence) as described in “Materials and Methods.” Blue flu-
orescence represents the nuclei stained with TOTO-3. A and A’ : inuntreated cells, the EGF-R was localized primarily on the cell surface;no WHI-P154-like immunoreactivity was detected. B and B’: after a30-mm incubation with unconjugated WHI-P154, no change in EGF-Rimmunoreactivity was detected (B). No WHI-P154 immunoreactivity
was detected in the cells (B’). C and C’ : after a 10-mm exposure,
EGF-P154 was bound to the cell surface EGF-R (arrowheads) and
started internalizing into the cytoplasm. D and D’ : by 30 mm, most of
the EGF-R/EGF-Pl54 complexes were internalized and deposited in theperinuclear region (arrows).
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
WHI-Pi 54:24hr� ..� .
� ��... .s,
A
EGF-P1 54:24hr
�� ; .� �,.
�B B’
..- U87/EGF�P154
..- U87!WHI�P154
.‘- U87/EGF
.6- U87/EGF.GEN
�0- U373IEGF.P154
.0- U373IWHI�P154
-4- U373/EGF
-.. WHI.P154-6- EGF.P154
N
CC>
C/)
ci�025
10 100
B. NALM-6 cells
10001
Clinical Cancer Research 1411
Fig. 4 Uptake of WHI-P154 and EGF-P154 by U373 human glioblastoma cells.
U373 cells were incubated with unconju-
gated WHI-P154 (A and A’) or EGF-Pl54(5 p.g/ml; B and B’) for 24 h and processedfor immunofluorescence to detect EGF-Rmolecules using a monoclonal antibody tothe EGF-R (green fluorescence) and a poly-clonal antibody to WHI-P154 (red fluores-cence) as described in “Materials andMethods.” Blue fluorescence represents thenuclei stained with TOTO-3.
010 100 1000
Drug Concentration ( �.tM)
Fig. 5 A, cytotoxic activity of EGF-P154 against brain tumor cells. U373 and U87 glioblastoma cells were incubated with EGF, EGF-Pb54,EGF-GEN, or unconjugated WHI-P154 for 36 h in 96-well plates, and the cytotoxicity was determined by the MiT assay. The data points represent
the means from three independent experiments; bars, SE. The mean IC50s for EGF-P154 against U373 and U87 cells were 0.813 ± 0.139 p.M and0.620 ± 0.097 p.M. respectively. The mean IC50s for unconjugated WHI-P154 against U373 and U87 cells were 167.4 ± 26.9 p.M and 178.6 ± 18.5
p.M, respectively. B, cytotoxic activity of unconjugated WHI-Pl54 and EGF-P154 on EGF-R-negative leukemia cells. Nalm-6 cells were incubated
with unconjugated WHI-P154 or EGF-P154 for 36 h in 96-well plates, and the cell survival was determined by the MU assay. Unconjugated
WHI-Pl54 showed considerable cytotoxic activity on Nalm-6 cells (IC50, 9.1 ± 1.2 p.M), whereas EGF-Pl54 showed no cytotoxicity. The data points
represent the means from three independent experiments; bars, SE.
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Control EGF
..� p’�..:‘�;:‘�., j...
EGF-P1 54
i,
‘�..�_:4._,�,- ,,�
A B
j’,:
s;��::��
C
EGF-P1 54
‘*�;�
0
fr
:. ,� � ,�,,*
�
, �,. .
.. .
e.
,
�.
,f� -
�,
. -!�, -4*�
‘S#{149}
�
*:�:�
�#{248}
-�
.-i
�,. --
1*! .�H
.� . � “e
�.
.-� � S t��P
-�, 4 --p!�r�’ � �
. �, �i.� .. - � .� -3�, 4:
- . � #{149}�.#{149}*
D� t:
�v �
2,! �. �:fr
5!
*�
- ,�4 :
a.-r� -
.j�
�- ‘ �..
1412 Activity of WHI-P154 against Human Glioblastoma Cells
Fig. 6 EGF-Pl54 induced apoptosis in brain tumor cells. A-C, U373 cells were incubated with 25 p.g/mb of EGF or EGF-Pl54 for 24 h and processedfor immunofluorescence using a monoclonal antibody to a-tubulin (green fluorescence). EGF-Pl 54 (but not EGF)-treated cells showed markedshrinkage with disruption of microtubules and lost their ability to adhere to the substratum. Blue fluorescence represents nuclei stained with TOTO-3.D-E, U373 cells were incubated with 25 p.g/ml of EGF-P154 for 36 h, processed for the in situ apoptosis assay, and analyzed with a laser scanningconfocab microscope. When compared with controls treated with EGF (D), several of the cells incubated with EGF-Pl54 (E) showed apoptotic nuclei
(yellow fluorescence). Red fluorescence represents nuclei stained with propidium iodide.
using immunofluorescence and confocal laser microscopy for
the internalized EGF-R and EGF-P154 molecules as well as for
evaluating the morphological changes in treated cells. EGF-
P154, similar to unconjugated EGF (not shown), was able to
bind to and enter target glioblastoma cells via receptor-mediated
endocytosis by inducing internalization of the EGF-R mole-
cubes. Within 10 mm after exposure to EGF-Pl54, the EGF-RJ
EGF-Pl54 complexes began being internalized, as determined
by cobocalization of the EGF-R (detected by anti-EGF-R anti-
body, green fluorescence) and EGF-Pl54 (detected by anti-P154
antibody, red fluorescence) in the cytoplasm of treated cells
(Fig. 3). By 30 mm, the internalized EGF-RIEGF-P154 com-
plexes were detected in the perinuclear region of the treated
glioblastoma cells. In contrast, cells treated with unconjugated
WHI-P154 alone did not reveal any detectable redistribution of
the surface EGF-R or cytoplasmic staining with the anti-P154
antibody (i.e., red fluorescence). By 24 h, WHI-P154 molecules
could also be detected in cells treated with unconjugated WH1-
P154 (Fig. 4). Thus, in accordance with our expectation, con-
jugation of WHI-P154 to EGF resulted in increased and more
rapid uptake of this cytotoxic quinazoline derivative by EGF-
R-positive glioblastoma cells.
We next sought to determine whether the improved deliv-
ery of WHI-Pl54 to glioblastoma cells by conjugation to EGF
results in potentiation of its antitumor activity. To this end, we
compared the cytotoxic activities of EGF-Pl54 and unconju-
gated WHI-Pl54 against U373 and U87 human glioblastoma
cell lines in dose-response studies using in vitro MU assays. As
shown in Fig. SA, EGF-Pl54 killed these glioblastoma cells in
each of three independent experiments at nanomolar concentra-
tions with mean IC50s of 813 ± 139 flM (range, 588-950 nM) for
U373 cells and 620 ± 97 nM (range, 487-761 nM) for U87 cells.
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
700
600
�50o
EaE� 4000>0
�300
200
100
-.- PBS
-0- EGF-‘.. WHI.P154 (1 mg/kg/d)
-.- EGF.P154 (0.5 mg/kg/d)
-0- EGF’P154(1 mg/kg/d)
A
a>
U)aa
L�.
0E
I-
-0- Control
-4-’WHI.P154 (1 mg/kg/d)
-0- EGF-P154 (0.5 mg/kg/d)
--EGF.P154(1 mg/kg/d)
10 20 30 40 50
Days After Inoculation60
Clinical Cancer Research 1413
The IC50s derived from the composite cell survival curves were
881 nsi for U373 cells and 601 nr�t for U87 cells. By comparison,
unconjugated WHI-Pl54 killed U373 or U87 cells only at
micromolar concentrations. EGF-P154 was 206-fold more po-
tent than unconjugated WHI-Pl54 against U373 cells (IC50s:
167.4 ± 26.9 p�M versus 81 1 ± 139 nM, P < 0.003) and
288-fold more potent than unconjugated WHI-P154 against U87
cells (IC50s: 178.62 ± 18.46 p.M versus 620 ± 97 nM, P <
0.001; Fig. SA). Unlike WHI-P154, which showed marked cy-
totoxicity against the EGF-R-negative NALM-6 leukemia cells,
EGF-P154 elicited selective cytotoxicity to EGF-R-positive
glioblastoma cell lines only (Fig. 5). Thus, conjugation to EGF
increased the potency of WHI-P154 against human glioblastoma
and at the same time restricted its cytotoxicity to EGF-R-
positive targets. Unlike the EGF-Pl54 conjugate, EGF-GEN, a
potent inhibitor of the EGF-R tyrosine kinase and EGF-R-
associated Src family PTK (22, 23), failed to kill glioblastoma
cells (Fig. SA). Thus, the potent cytotoxicity of EGF-Pl54
cannot be explained by the tyrosine kinase inhibitory properties
of its WFII-Pl54 moiety.
We next used immunofluorescence staining with anti-a-
tubulin antibody and the nuclear dye TOTO-3 in combination
with confocal baser scanning microscopy to examine the mor-
phobogical features of U373 glioma cells treated with either
unconjugated EGF or EGF-P154. After 24 h of exposure to 25
jig/mI EGF-Pl 54 (but not 25 p.g/ml unconjugated EGF), most
of the glioma cells showed an abnormal architecture with com-
plete disruption of microtubules, marked shrinkage, nuclear
fragmentation, and inability to adhere to the substratum (Fig. 6,
A-C). These morphological changes in EGF-P154-treated gli-
oma cells were consistent with apoptosis. To confirm apoptotic
DNA fragmentation in the nuclei of EGF-Pl54-treated glioblas-
toma cells, we used an in situ apoptosis assay that allows the
detection of exposed 3’-hydroxyl groups in fragmented DNA by
TdT-mediated dUTP nick-end labeling. As evidenced by the
confocal laser scanning microscopy images depicted in Fig. 6, D
and E, EGF-Pl54-treated (but not EGF-treated) glioma cells
examined for digoxigenin-dUTP incorporation using FITC-con-
jugated anti-digoxigenin (green fluorescence) and propidium
iodide counterstaining (red fluorescence) showed many apop-
totic yellow nuclei with superimposed green and red fluores-
cence at 36 h after treatment.
In Vivo Antitumor Activity of EGF-P154 in a SCID
Mouse Xenograft Model of Human Glioblastoma. CB.17
SCID mice develop rapidly growing tumors after s.c. inoculation of
0.5 x 106 U373 cells. We examined the in vivo antitumor activity
of EGF-Pl54 in this SCID mouse xenograft model of human
glioblastoma multiforme. EGF-Pl54 significantly improved tumor-
free survival in a dose-dependent fashion, when it was administered
24 h after inoculation oftumor cells. Fig. 7 shows the tumor growth
and tumor-free survival outcome of SCID mice treated with EGF-
P154 (500 �Lg/kg/day X 10 days or 1 mg/kg/day X 10 days),
unconjugated EGF (1 mg/kg/day X 10 days), unconjugated WHI-
P154 (1 mg/kg/day X 10 days), or PBS after inoculation with U373
glioblastoma cells. None of the 15 control mice treated with PBS
(n 5; median tumor-free survival, 19 days), EGF (n = 5; median
tumor-free survival, 23 days), or unconjugated WHI-P154 (n = 5;
median tumor-free survival, 19 days) remained alive tumor-free
beyond 33 days (median tumor-free survival, 19 days; Fig. 7A). All
0 10 20 30 40 50 60 70
Days After Inoculation
Fig. 7 In vivo antitumor activity of EGF-P154 against human glioblas-toma in SCID mice. Twenty-four h after s.c. inoculation of U373
glioblastoma cells, mice were treated with EGF-P154 (0.5 mg/kg/day X
10 days, n - 5, or I .0 mg/kg/day X 10 days, n = 5), EGF (1.0mg/kg/day x 10 days, n = 5), unconjugated WHI-Pl54 (1.0 mg/kg/day X 10 days, n 5), or PBS (n = 5). Mice were monitored for healthstatus and tumor growth. A compares the effect of these treatments on
tumor growth and progression. B depicts the tumor-free survival curves
from SCID mice treated with PBS or EGF (Control: n 10), uncon-
jugated WHI-P154 (n 5), or EGF-P154 (n = S for each of the twodose levels tested). Comparisons of outcome between groups were doneusing the log-rank test. *, no measurable tumors were observed.
of the five mice treated with EGF-P154 at the 500 p.g/kg/day dose
level developed tumors within 40 days with an improved median
tumor-free survival of 33 days (Fig. 7B), and the tumors were much
smaller than in control mice (Fig. 7A). Tumors reached a size of 50
mm3 by 37.5 ± 3.3 days in PBS-treated mice, 34.0 ± 3.0 days in
EGF-treated mice, 36.0 ± 5.1 days in WHI-Pb54-treated mice.
Tumors developing in EGF-Pl54 (500 p.g/kg/day X 10 days)-
treated mice reached the 50-mm3 tumor size -� I 1 days later than
the tumors in control mice treated with PBS, EGF, or WHI-Pl54
(47.4 ± 7. 1 days versus 35.8 ± I .8 days). The average sizes
(mean ± SE) of tumors at 20 and 40 days were 10.2 ± 1.4 mm3
and 92.3 ± 6.0 mm3, respectively, for mice in the control group
(i.e., PBS + EGF groups combined). By comparison, the average
sizes (mean ± SE) of tumors at 20 and 40 days were significantly
Research. on March 24, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1414 Activity of WHI-Pl54 against Human Glioblastoma Cells
smaller at 1 .0 ± 1 . I mm3 (P = 0.002) and 37.6 ± 10.7 mm3 (P =
0.0003) for mice treated with EGF-P154 at the 500-p.g/kg/day dose
level. Notably, 40% of mice treated for 10 consecutive days with 1
mg/kg/day EGF-P154 remained alive and free ofdetectable tumors
for >58 days (PBS + EGF + WHI-Pl54 versus EGF-Pl54, P <
0.00001 by log-rank test). The tumors developing in the remaining
60% of the mice did not reach a size >50 mm3 during the 58-day
observation period. Thus, EGF-Pl54 elicited significant in vivo
antitumor activity at the applied nontoxic dose levels. The inability
of 1 mg/kg/day x 10 days of unconjugated WHI-Pl54 (53.2 nmol)
and unconjugated EGF to confer tumor-free survival in this SCID
mouse model in contrast to the potency of 1 mg/kg/day X 10 days
of EGF-P154 (corresponding to 2.9 nmol of WFII-P154) demon-
strates that: (a) the in vivo antitumor activity of EGF-Pl54 cannot
be attributed to its EGF moiety alone; and (b) conjugation to EGF
enhances the in vivo antitumor activity of WFII-P154 against glio-
blastoma cells by >18-fold.
Taken together, our findings provide unprecedented evi-
dence that WHI-P154 exhibits significant cytotoxicity against
human glioblastoma cells, and its antitumor activity can be
substantially enhanced by conjugation to EGF as a targeting
molecule. Although WHI-P154 is a potent inhibitor of the
EGF-R kinase as well as Src family tyrosine kinases, its cyto-
toxicity in glioblastoma cells cannot be attributed to its tyrosine
kinase inhibitory properties alone, because WHI-P79 with
equally potent PTK inhibitory activity failed to kill WHI-P154-
sensitive glioblastoma cells. Similarly, several PTK inhibitors
capable of killing human leukemia and breast cancer cells
lacked detectable cytotoxicity against glioblastoma cells. Glio-
bbastoma cells exposed to EGF-conjugated WHI-P154 under-
went apoptosis. Future identification of the molecular target for
WHI-P154 may lead to a structure-based design of potentially
more active antiglioblastoma agents. Finally, although we used
EGF to target WHI-P154 to glioblastoma cells in the present
study, other biological agents, including different cytokines
such as insulin-like growth factor and antibodies reactive with
glioblastoma-associated antigens, may be equally effective or
better targeting molecules for this novel quinazoline derivative.
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1998;4:1405-1414. Clin Cancer Res R K Narla, X P Liu, D E Myers, et al. against human glioblastoma cells.a novel quinazoline derivative with potent cytotoxic activity 4-(3'-Bromo-4'hydroxylphenyl)-amino-6,7-dimethoxyquinazoline:
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