The Antiproliferative Effects of Tyrosine Kinase Inhibitors Tyrphostins on a Human Squamous Cell Carcinoma in Vitro and in Nude Mice1

Abstract

Many human tumors of epithelial origin contain cells overexpressing the epidermal growth factor (EGF) receptor, and there is convincing evidence that cancer cell growth is correlated with the loss of the normal regulation of the EGF receptor signal transduction pathway. Some cancers are clearly dependent on activation of the EGF receptor for their proliferation. Recently, a class of compounds, tyrphostins, which inhibit the protein tyrosine kinase activity of the growth factor receptor, have been described. In this report, we have examined the antiproliferative effects of potent new tyrphostins on a well-characterized human squamous cell carcinoma in vitro and in vivo. We found that two of these compounds (RG-13022 and RG-14620) suppressed not only EGF-stimulated cancer cell proliferation in vitro but also tumor growth in nude mice. RG-13022 also increased the life span of these tumor-bearing nude mice. When administered to tumor-bearing nude mice together with monoclonal antibodies to the EGF receptor at a suboptimal dose which had no effect alone, inhibition of tumor growth was markedly enhanced. These data suggest that tyrphostins have potential as anticancer agents.

Full text

[CANCER RESEARCH 51. 4430-4435, August 15, 1991]
The Antiproliferative Effects of Tyrosine Kinase Inhibitors Tyrphostins on a
Human Squamous Cell Carcinoma in Vitro and in Nude Mice1
Toshiyuki Yoneda,2 Ray M. Lyall, Maria M. Alsina, Paul E. Persons, Alfred P. Spada, Alexander Levitzki,
Asher Zilberstein, and Gregory R. Mundy
Division of Endocrinology and Metabolism, Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7877
¡T.Y., M. M. A., G. R. M.J; Rhône-Poulenc Rarer Central Research, Horsham, Pennsylvania 19044 [R. M. L., P. E. P., A. P. S., A. Z.]; and the Department of Biological
Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel [A. L.]
ABSTRACT
Many human tumors of epithelial origin contain cells overexpressing
the epidermal growth factor (EGF) receptor, and there is convincing
evidence that cancer cell growth is correlated with the loss of the normal
regulation of the EGF receptor signal transduction pathway. Some can
cers are clearly dependent on activation of the EGF receptor for their
proliferation. Recently, a class of compounds, tyrphostins, which inhibit
the protein tyrosine kinase activity of the growth factor receptor, have
been described. In this report, we have examined the antiproliferative
effects of potent new tyrphostins on a well-characterized human squa-
mous cell carcinoma in vitro and in vivo. We found that two of these
compounds (RG-13022 and RG-14620) suppressed not only EGF-stim-
ulated cancer cell proliferation in vitro but also tumor growth in nude
mice. RG-13022 also increased the life span of these tumor-bearing nude
mice. When administered to tumor-bearing nude mice together with
monoclonal antibodies to the EGF receptor at a suboptimal dose which
had no effect alone, inhibition of tumor growth was markedly enhanced.
These data suggest that tyrphostins have potential as anticancer agents.
INTRODUCTION
Human squamous cell carcinomas are among the most diffi
cult human malignancies to treat (1). They are notoriously
resistant to chemotherapy and radiation therapy, particularly
when they originate in the lungs, head, or neck (2, 3). Many
human squamous cell carcinomas contain large numbers of
EGF3 receptors (4) and may be dependent on the EGF receptor
signaling pathway for their proliferation in vitro and growth in
vivo (5, 6). Interfering with this pathway by pharmacological
means may provide an effective medical treatment for these
cancers.
In this paper, we describe the effects of several tyrphostins
on EGF-stimulated cell proliferation in tumor cells and on
EGF receptor tyrosine kinase activity in vitro. Two of these
compounds, RG-13022 and RG-14620, have longer-lasting ef
fects in cell culture than do the previously described tyrphostins
(7, 8), and this property prompted us to test these compounds
in vivo. We characterized the effects of these factors on cells
overexpressing EGF receptors to confirm their capacity to
inhibit EGF-dependent tyrosine kinase. These compounds were
administered to nude mice bearing the well-characterized hu
man squamous cell carcinoma MH-85, which is associated with
three paraneoplastic syndromes, hypercalcemia, leukocytosis,
Received 3/11/91; accepted 6/6/91.
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.
'This work was supported in part by Grants AR-28149, CA-40035, DE-
08569, and DE-08526 from the NIH.
2To whom requests for reprints should be addressed, at Division of Endocri
nology and Metabolism. Department of Medicine, The University of Texas
Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX
78284-7877.
3The abbreviations used are: EGF, epidermal growth factor; PCS, fetal calf
serum; DMSO, dimethyl sulfoxide; DMEM. Dulbecco's modified Eagle's me
dium; «MEM, a minimal essential medium; in Mil OS. monoclonal antibody to
human EGF receptor; ICM, concentration producing 50% inhibition.
and cachexia (9, 10). We found that RG-13022 and RG-14620
not only inhibited EGF-stimulated proliferation of MH-85 cells
in vitro but suppressed the growth of MH-85 tumors in nude
mice and the expression of the paraneoplastic syndromes. Tu
mor-bearing nude mice treated with these agents lived signifi
cantly longer than corresponding tumor-bearing mice treated
by injections of vehicle.
MATERIALS AND METHODS
Compounds
The tyrphostins used in this study were RG-13022 and RG-14620
(Fig. 1). Forty HIMstock solutions of these compounds were made in
100% DMSO and diluted with the culture medium before addition to
the cells. The culture medium containing the equivalent concentrations
of DMSO served as vehicle controls and had no effect on MH-85
growth in culture and in nude mice.
HER14 Cells
Cells termed HER14 were prepared by transfecting NIH 3T3 (clone
2.2) (from Charlotte Fryling, National Cancer Institute, NIH), which
lack endogenous EGF receptors, with complementary DNA constructs
of wild-type human EGF receptor, as described (11, 12). HER14 cells
were shown to express high intrinsic tyrosine kinase activity. HER 14
cells were grown in DMEM (Gibco Laboratory, Grand Island, NY)
containing 10% calf serum (HyClone Laboratories, Logan, UT) and
supplemented with 2 mM L-glutamine and 100 units/ml of penicillin-
streptomycin (Gibco).
MH-85 Tumor and Cells
MH-85 tumor was derived from a patient with a squamous cell
carcinoma of the maxilla who manifested marked leukocytosis
(180,000/mnr1; >90% mature granulocytes), cachexia (loss of body
weight, muscle, and adipose tissue), and hypercalcemia (13.0 mg/dl)
(9). MH-85 cells were established in culture from the tumors formed
in athymic nude mice (10). The cells were grown in «MEM(Hazleton
Biologies, Inc., Lenexa, KA) supplemented with 10% PCS (HyClone)
and 1% penicillin-streptomycin solution.
Cell-free Autophosphorylation of EGF Receptor in Immunoprecipitates
Confluent HER 14 cell layers from 15-cm tissue culture dishes were
solubili/ed by scraping the cells in 1 ml of cell lysis buffer (50 mM 4-
(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.5-150 mM
NaCl-1.5 mM MgCl2-l mM EGTA-10% glycerol-1% Triton X-100-4
Mg/ml phenylmethylsulfonyl fluoride-1 Mg/ml aprotinin-1 Mg/ml leu-
peptin). The lysates were cleared by centrifuging at maximum speed in
an Eppendorf microcentrifuge for 15 min at 4°C.Fifty ti\ of lysate were
used for each reaction. EGF (0.5 Mg/ml; Toyobo Inc., New York, NY)
was added to the lysate for 10 min at 4°Cprior to immunoprecipitation.
Lysates were incubated with protein A-Sepharose-purified mAblOS
(13) complex for 90 min in the cold and then washed 3 times with
buffer (20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH
7.5-150 mM NaCl-0.1% Triton X-100-10% glycerol). The washed im-
munoprecipitate was aliquoted and preincubated for 30 min on ice with
tyrphostins in 30 M'of the same buffer containing 5 mM MnCl2 and
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RG 14620
TYROSINE KINASE INHIBITOR ON HUMAN SQUAMOUS CANCER GROWTH
RG 13022
M.W. 275
C.6H,,NSO,
M.W. 266
Fig. 1. Chemical structures of RG-14620 (left) and 13022 (right).
200 nM sodium orthovanadate. Autophosphorylation was initiated by
adding 4.5 nC\ of [7-32P]ATP (Dupont NEN, Wilmington, DE) and
unlabeled ATP to give a final concentration of 2 ^M ATP. After 2 min
at 4°Cthe reaction was terminated with 55 ^1 of 2x sodium dodecyl
sulfate sample buffer, and the samples were electrophoretically sepa
rated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel (14). Quan-
titation was carried out by densitometry scanning of the
autoradiograms.
Autophosphorylation of EGF Receptor in HER14 Cells
Confluent HER 14 cells in 10-cm tissue culture dishes were trans
ferred to DMEM containing 0.5% PCS and incubated with tyrphostins
either overnight or for various periods of time. The cells were then
stimulated for 5 min with EGF (500 ng/ml) and lysed and subjected to
immunoblotting with anti-phosphotyrosine antibodies as described
(15). Quantitation was carried out by densitometry scanning of
autoradiograms.
Colony Formation
MH-85 cells (100/well; 24-well plate; Corning Glassware, Corning,
NY) and HER 14 cells (200/dish; 10-cm dish) were plated in complete
medium, either «MEMor DMEM, respectively, supplemented with
10% FCS. After overnight culture, the culture medium was switched to
«MEMsupplemented with 0.2% PCS and 50 ng/ml EGF (MH-85) or
DMEM supplemented with 0.5% PCS and 50 ng/ml EGF (HER14).
The cells were cultured in this medium in the presence or absence of
increasing concentrations of RG-13022 or RG-14620 for 10 days. At
the end of culture, the cells were fixed with 4% (v/v) formaldehyde in
calcium-magnesium-free phosphate-buffered saline for 15 min at room
temperature and stained with hematoxylin (13). Numbers of colonies
including more than 20 cells in each well were counted under the
microscope.
DNA Synthesis
MH-85. MH-85 cells (1 x 10"/0.1 ml) were plated onto 96-well
plates (fiat bottom; Corning) and cultured in «MEM supplemented
with 10% PCS overnight. The cells were then fed with «MEMsupple
mented with 0.2% PCS and 50 ng/ml EGF and cultured in this medium
in the absence or presence of increasing concentrations of RG-13022
or RG-14620 for 44 h. The cells were then incubated with 0.2 MCi/well
[me/A>>/-3H]thymidine (2.0 Ci/mmol; ICN Radiochemicals, Costa
Mesa, CA) for a further 4 h. At the end of incubation, the cells were
detached by treatment with trypsin-EDTA solution (Gibco) and har
vested onto glass fiber strips using a multiple automated cell harvester
(Cambridge Technology, Inc., Watertown, MA). The radioactivity was
measured by liquid scintillation counting.
HER14 Cells. DNA synthesis in HER14 cells was measured as
described (8)
Inoculation of MH-85 Tumor into Nude Mice
MH-85 tumors 5 mm in diameter were inoculated s.c. into the right
dorsal portion of 4- to 6-week-old male BALB/c nu/nu mice (HarÃ-an
Sprague-Dawley, Indianapolis, IN). Animals were fed ad libitum on
autoclaved diet (Tek-land-LH-485; calcium, 0.83 mg/kg; phosphorus,
0.65 mg/kg; HarÃ-anSprague-Dawley) and tap water (acidified to pH
2.5). Tumor sizes were measured once a week under anesthesia with
nembutal (0.05 mg/g body weight, i.p.) and calculated as described
(16).
Administrationof Tyrphostins in MH-85 Tumor-bearingNude Mice
RG-13022 or RG-14620 in 0.1 ml 100% DMSO was injected i.p.
twice a day from 1day after MH-85 tumor inoculation. Control animals
were given the same vehicle.
RESULTS
Inhibition of Cell-free EGF Receptor Autophosphorylation by
RG-13022. In a cell-free reaction RG-13022 inhibited the au-
tophosphorylation reaction of the EGF receptor in immuno-
precipitates with an IC50of 4 /UM(Fig. 2). An amount of DMSO
equivalent to that used in the highest RG-13022 dose showed
no inhibitory effect (data not shown).
Inhibition of EGF Receptor Autophosphorylation in Intact
HER14 Cells by RG-13022. RG-13022 inhibited autophos-
phorylation of the EGF-receptor in HER 14 cells after an over
night incubation (Fig. 3/1). The IC50 of RG-13022 was 5 MM.
When a maximally inhibitory concentration of RG-13022 (60
MM)was added to HER14 cells and the incubation was contin
ued for various times, it was found that maximal inhibition of
autophosphorylation persisted even up to 48 h (Fig. 3ß).This
was in contrast to the situation found for RG-50864 (8) for
which inhibition was reversed by 48 h.
Inhibition of HER14 Growth in Culture by RG-13022 and
RG-14620. RG-13022 and RG-14620 inhibited colony forma
tion and DNA synthesis by HER 14 cells, which were stimulated
by 50 ng/ml EGF, in a dose-dependent manner (Fig. 4). The
ICsoSfor RG-13022 and RG-14620 were 1and 3 MMfor HER 14
colony formation and 3 and 1 pM for HER 14 DNA synthesis,
respectively.
The longer-lasting actions of RG-13022 and RG-14620 on
autophosphorylation and growth in HER 14 cells in vitro led us
to examine these compounds in MH-85 tumors in vivo as well
as on MH-85 cells in vitro. MH-85 cells were shown to over-
express endogenous EGF receptors and are dependent on the
o
C
Q.
M
O
C.
o.
o
I
10 I
20
[Tyrphostin] uM
Fig. 2. Effect of RG-13022 on cell-free autophosphorylation of the EGF
receptor in immunoprecipitates. Washed immunoprecipitates of EGF receptor
were preincubated with the indicated concentrations of RG-13022 for 30 min.
Autophosphorylation was initiated by adding [>-32P]ATP, and the reaction was
terminated after 2 min by adding sodium dodecyl sulfate sample buffer. Inset.
results of densitometric scanning of the autoradiogram. The same results were
obtained in several separate experiments.
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TYROSINE KINASE INHIBITOR ON HUMAN SQUAMOUS CANCER GROWTH
EGF receptor signal transduction pathway for their growth
both in vitro and in nude mice (17).
Inhibition of Ml 1-85 Growth in Culture by RG-13022 and RG-
14620. RG-13022 and RG-14620 also suppressed colony for
mation and DNA synthesis by EGF-stimulated MH-85 cells
(Fig. 5) in a dose-dependent manner. The IC50s for RG-13022
and RG-14620 on MH-85 cells were 7 and 4 /¿Mfor colony
formation and 1.5 and 1.25 /¿Mfor DNA synthesis, respectively.
The growth-inhibitory effect of RG-13022 and RG-14620 was
reversible (data not shown). Growth of Chinese hamster ovary
cells (18), which do not express EGF receptors, was not signif
icantly decreased by RG-13022 even at concentrations as high
as 40 /¿M(data not shown).
Inhibition of MH-85 Growth in Nude Mice by RG-13022 and
RG-14620. Nude mice implanted with MH-85 tumors mani
fested profound hypercalcemia, cachexia, and leukocytosis as
the tumor grew (Fig. 6, right; Ref. 10). RG-13022 (400 Mg/
mouse/day) significantly inhibited MH-85 tumor growth (Fig.
7). Because of slower tumor growth, MH-85 tumor-bearing
animals receiving injections of RG-13022 showed less cachexia
and hypercalcemia (Fig. 6, left), ate more food, and were more
active than untreated MH-85 tumor-bearing animals. As a
o
£
O.
t/1
O
-C
O.
o
—r—
20
[Tyrphostin]
B
*
o.
in
o
O.
o
100
50 -
20 I
40
Time (hours)
•RG13022
* RG14620
[Tyrphostin] uM
B
Fig. 3. Effect of RG-13022 on autophosphorylation of the EGF receptor in
HERI4 cells. In A, cells were incubated for 17 h with RG-13022, stimulated for
5 min with EGF, lysed, and subjected to immunoblotting with anti-phosphoty-
rosine antibodies. H. kinetics. Cells were incubated with 60 (/\i RG-13022 (•)
and 100 UM RG-50864 (•).The same results were obtained in several separate
experiments.
4432
[Tyrphostin] uM
Fig. 4. Effect of RG-13022 (•)and RG-14620 (A) on colony formation (A)
and DNA synthesis (A) by HER14 cells. Cells (200-100-mm dish in A and 1 x
lO'/well, 24 wells in B) were treated with RG-13022 or RG-14620 in the presence
of 50 ng/ml EGF for 10 days (A) and 17 h (B). Assays were carried out in
duplicate. The same results were obtained in several separate experiments.
result of suppression of tumor growth, RG-13022 prolonged
the life span of MH-85 tumor-bearing animals (Fig. 8). Admin
istration of RG-13022 at a dose of 100 Mg/mouse/day did not
show any effect on MH-85 tumor growth (Fig. 9). A newer
compound, RG-14620, at a dose of 200 ^g/mouse/day inhibited
MH-85 tumor growth in nude mice to the same degree as that
caused by 400 ßgRG-13022 (Table 1).
Effect of RG-13022 and inAblOS Combination on MH-85
Growth in Nude Mice. We previously showed that MH-85
growth in nude mice was markedly suppressed by anti-EGF
receptor mAblOS (17). Since mAb 108 and tyrphostins act at
different steps in the signaling pathway, we thought it possible
that the effects of RG-13022 may be enhanced if used in
conjunction with mAb 108. Administration of mAblOS i.p. (1
mg/mouse/day) at 1, 5, and 10 days after MH-85 inoculation
of RG-13022 (400 /¿g/mouse/day) for 14 days profoundly in
hibited MH-85 growth in nude mice (Fig. 9). Lower concentra
tions of mAblOS (10 Mg) or RG-13022 (100 ^g) failed to
decrease MH-85 growth. However, combined administration
of these lower concentrations of mAblOS and RG-13022 syn-
ergistically decreased MH-85 growth (Fig. 9).
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TYROSINE KINASE INHIBITOR ON HUMAN SQUAMOUS CANCER GROWTH
Colony Formation
•RG 13022
* RG 14620
10 20
Tyrphostin
100» 3H-TdR Incorporation
30 40
•RG 13022
A RG 14620
40
20
"'it)
10 2030Tyrphostin
(iM40
Fig. 5. Effect of RG-13022 (•)or RG-14620 (A) on colony formation (A) and
DNA synthesis (B) by MH-85 cells. Cells (100/well, 24 wells in A and 1 x IO4/
well, 96 wells in B) were treated with RG-13022 or RG-14620 in the presence of
50 ng/ml EGF for 10 days (A) and 48 h (B). Colony numbers and [3H]dThd
uptake by EGF-stimulated MH-85 cells which were not treated with RG-13022
and RG-14620 were 48/well and 18,322 dpm/well, respectively. Assays were
carried out in duplicate. The same results were obtained in several separate
experiments. Ordinate, percentage of untreated cultures.
DISCUSSION
In this paper, we have shown that the tyrosine kinase inhibi
tors RG-13022 and RG-14620 inhibit tumor growth both in
vitro and in vivo. These compounds were found to inhibit EGF
receptor autophosphorylation in immunoprecipitates and in
intact living cells that overexpress the EGF receptor (HER 14).
In contrast to the previously described tyrosine kinase inhibitor
RG-50864 (8), these compounds continue to inhibit autophos
phorylation in intact cells even after 48 h in cell culture (Fig
3B). RG-13022 in solution undergoes a light-induced trans/cis
isomerization. The cis and trans forms of RG-13022 are equally
inhibitory, and their biological effect is stable in cell culture for
many days.4 In contrast, RG-14620 is chemically stable. It was
this property of the prolonged duration of intracellular effect
that suggested RG-13022 and RG-14620 as potential antican-
cer candidates in vivo. RG-13022 and RG-14620 should be
effective in those tumors which contain cells which overexpress
the EGF receptor and respond to inhibition of the EGF receptor
signaling pathway. We assessed the effects of these agents on
the growth of a human squamous cell carcinoma (MH-85)
which overexpresses in EGF receptors. In other experiments,
we have found that growth of this tumor in vivocan be inhibited
by anti-EGF receptor mAb 108 (3) and by removal of the sub-
mandibular glands which in the adult male mouse are the source
of more than 95% of the circulating EGF. Injection of EGF in
sialoadenectomized mice leads to enhanced MH-85 tumor
growth (17). Thus, growth of the MH-85 tumor in vivo is
dependent on the EGF receptor signaling pathway, and this
model provides a suitable system for examining the potential
effects of tyrphostins. Using this model, we have clearly dem
onstrated that two of the tyrphostin compounds (RG-13022
and RG-14620) suppress MH-85 tumor cell growth in culture
and in nude mice and prolong the survival of tumor-bearing
animals.
It is suggested that RG-13022 and RG-14620 are effective
when used from the time of tumor inoculation. When the
compounds were administered to animals after tumors were
already established they failed to cause significant tumor regres
sion.4 The same result is seen when monoclonal antibodies to
human EGF receptors (19) or other chemotherapeutic agents
(20) are used in established tumors. These agents all cause
cytotoxicity of a fraction of the proliferating tumor cells, and
thus the larger the tumor at the time of the administration, the
4Unpublished observations.
Fig. 6. Photograph of MH-85 tumor-bearing nude mice treated with (left) or
without (right) RG-13022. RG-13022 was administered i.p. from 1 day after
MH-85 tumor implantation for 14 days at a dose of 100 «ig/mouse/injection, 2
injections a day. The photograph was taken of the most representative animals
50 days after tumor implantation. Note the smaller tumor and less decrease in
body size, muscle, and adipose tissue in nude mouse treated with RG-13022 (left).
Blood ionized calcium was lower in the RG-13022-treated animal (1.62 mmol/
liter; left) than in the untreated animal (2.61 mmol/liter; right). Normal value of
blood ionized calcium is 0.95-1.25 mmol/liter.
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TVROSINE KINASE INHIBITOR ON HUMAN SQUAMOUS CANCER GROWTH
50-,
40-
E
A 30
o 20
10-
BG 13022
400 u,g/mouse/day (ij)
-«.mm
10 20 30 40 50
Days After Tumor Implantation
Fig. 7. Effect of RG-13022 on MH-85 tumor growth in nude mice. Arrows,
200 (jg/mouse/injection, 2 injections/day of RG-13022 were administered i.p.
from 1 day after MH-85 tumor implantation for 10 days (•).Control animals
(O) received equal amounts of vehicle. Points, mean of 3 separate experiments;
bars, SE; numbers in parentheses, number of animals studied: ', significantly
smaller than control group (P < 0.01 ).
400 lag/mouse/day, i.p.
a
E
5 6H
0)
CO 2-
RG13022
DMSO
:
10 12 16 18 20 22
Weeks After MH-85 Tumor Implantation
Fig. 8. Survival of MH-85 tumor-bearing animals treated with ( )or without
( ) RG-13022 (200 Mg/mouse/injection; 2 injections/day) every day for 5
weeks.
less the absolute effect. This suggests that these agents may be
most useful therapeutically if administered in conjunction with
a debulking treatment such as surgical ablation. Based on the
demonstration that combination of suboptimal doses of
mAb 108 and RG-13022 synergistically decreased MH-85
growth in nude mice, it is also possible that RG-13022 and
RG-14620 will have greater utility when used in conjunction
with drugs such as cisplatinum and doxorubicin. These agents
have been found to exert profound antitumor effects when used
in conjunction with anti-EGF receptor m Ab 108 in several
models of human cancer (13, 21, 22).
The MH-85 tumor is associated with three paraneoplastic
syndromes both in the original patient (9) as well as in tumor-
bearing nude mice (10). These paraneoplastic syndromes are
hypercalcemia, leukocytosis, and cachexia. Each of these syn
dromes was readily monitored in tumor-bearing mice by follow
ing blood ionized calcium, WBC, and body weight. When tumor
mass or burden was diminished by treatment with the tyrphos-
tins, we found a simultaneous decrease in the severity of these
paraneoplastic syndromes. Other experiments suggest these
syndromes are mediated, at least in part, by a tumor factor
which stimulates the production of cytokines such as tumor
necrosis factor by host immune cells, and these tumor products
and host cytokines work in concert to cause the paraneoplastic
syndromes (23). However, these syndromes are entirely de
pendent on the mass of the transplanted tumor, and decreasing
tumor mass by use of the tyrosine kinase inhibitors RG-13022
and RG-14620 also leads to abatement of the paraneoplastic
syndromes.
Our data suggest that tyrphostins may be useful anticancer
drug candidates if they can be shown to have limited or accept
able toxicity. Because of the ubiquitous presence of tyrosine
kinases in various normal tissues, the tyrosine kinase inhibitors
may be cytotoxic to these tissues as well as to MH-85 tumors.
However, in our experiments, growth of Chinese hamster ovary
cells which lack endogenous EGF receptors was not suppressed
by tyrphostins at concentrations 20 to 30 times higher than the
ICso for MH-85, suggesting that tyrphostins might act prefer
entially on cells with high intrinsic expression of EGF receptor
tyrosine kinase. Moreover, MH-85 tumor-bearing nude mice
treated with 400 ¿ig/dayRG-13022 or with 200 ng/day RG-
14620 for 4 weeks were more active, ate more food, and lost
less weight than untreated animals. This is partly due to de
creased tumor growth, but it at least suggests that RG-13022
and RG-14620 administered according to our protocol are not
Tumor Size (cm3) (Mean ?S.E., n = 6)
40130-20-
10-
0T';"!None*TIi1mAb108 RG130221mglOng 400iig 100|ig
t
*
mAb108 10iig
+ RG13022
100|ig
Fig. 9. Effects of RG-13022 and mAblOS on MH-85 tumor growth in nude
mice. RG-13022 (50 Kgor 200 ng/mouse/injection; 2 injections/day) was admin
istered i.p. from 1 day after MH-85 tumor inoculation for 14 days, m Ab lOK(10
ng or 1 mg/mouse/injection) was administered i.p. at days 1, 5, and 10 after MH-
85 tumor inoculation. Tumor size was measured 7 weeks after MH-85 tumor
inoculation. Columns, mean; bars, SE; n = 6. ", significantly smaller than untreated
control group (/' < 0.01): ', significantly smaller than animals treated with 10 Mg
mAblOS or 100 ^g RG-13022 alone (/>< 0.01).
Table 1 Effect of tyrophostins on MH-85 growth in nude mice
Tyrophostins (200 ng for RG-13022 or 100 ^g for RG-14620/mouse/injection;
2 injections a day) were injected i.p. from the time of tumor inoculation for 4
weeks. Tumor size was measured 5 weeks after tumor inoculation and calculated
as described in the text.
Treatment
(iig/mouse/day)DMSORG-13022
(400„g/mouse/day)RG-14620
(200 ^g/mouse/day)Mice1234512341234Tumor
size
(cm3)224420191831411.477105Mean
±SE25
±55
±3°7±
1"
1Significantly different from DMSO-treated group (/>< 0.01).
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TYROSINE KINASE INHIBITOR ON HUMAN SQUAMOUS CANCER GROWTH
harmful to the host animals. Of course, extensive histological
examination of the organs of animals receiving injections of
tyrphostins is needed.
Although the results described here have demonstrated that
tyrosine kinase inhibitor tyrphostins inhibit EGF-dependent
growth of MH-85, our more recent data have shown that
tyrphostins also suppress in vitro growth of human breast cancer
cells such as MCF-7 and T47D cells which are known to express
relatively low levels of EGF receptors (24). Furthermore, tyr
phostins inhibit growth of breast cancer cell which is stimulated
by insulin, insulin-like growth factor I and II, and estrogen (25).
Thus, antiproliferative effects of tyrphostins are not as specific
for cells with overexpression of EGF receptors as we had
initially anticipated but do seem to be effective on malignant
cells with high intrinsic tyrosine kinase activity.
The effects of tyrphostins on other non-EGF receptor types
of tyrosine kinase have not been extensively studied yet. In our
preliminary experiments, RG-13022 decreased the activity of
tyrosine kinase in human osteosarcoma cell line MG-63 which
shows the same mobility as that of src tyrosine kinase of the
human leukemia cell line HL-60 (26) on a native gel,4 suggest
ing tyrphostins act also on non-receptor-type tyrosine kinases
such as src.
In conclusion, this work demonstrates that the compounds
which inhibit the tyrosine kinase activity of the EGF receptor
also inhibit the growth of a human squamous cell carcinoma in
nude mice. These results raise the possibility that tyrosine
kinase inhibitors may prove to be useful agents for the treatment
of a variety of cancers in which tyrosine phosphorylation by
oncogene products plays a role in cell transformation.
ACKNOWLEDGMENTS
We are thankful to Drs. J. Schlessinger, J. A. Haimovich, and A. B.
Schreiber for their fruitful discussions and to Nancy Garrett, R.
Ratkiewicz, and Thelma Barrios for their expert secretarial assistance
in the preparation of the manuscript.
REFERENCES
1. Silberberg, E. Cancer statistics. CA Cancer J. Clin., 3S: 19-35, 1985.
2. Merino, O., Lindberg, R. D., and Fletcher, G. H. An analysis of distant
métastasesfrom squamous cell carcinoma of the upper aero-digestive tract.
Cancer (Phila.), 40:145-151, 1977.
3. Vikram, B. Changing patterns of failure in advanced head and neck cancer.
Arch. Otolaryngol., 110: 564-565, 1984.
4. Hendler, F., Simm. A., Richards,C., Cassels, D., Guasterson, B., and Ozanne,
B. Evidence for increased epidermal growth factor receptors in epidermal
malignancies. Cancer Cells (Cold Spring Harbor), /: 41-50, 1984.
5. Carpenter, G. Receptors for epidermal growth factor and other polypeptide
mitogens. Annu. Rev. Biochem., 56: 881-914, 1987.
6. Ullrich, A., and Schlessinger, J. Signal transduction by receptors with tyro
sine kinase activity. Cell, 61: 203-212, 1990.
7. Yaish, P., Gazit, A., Gilon, C, and Levitzki, A. Blocking of EGF-dependent
cell proliferation by EGF receptor kinase inhibitors. Science (Washington
DC), 242: 933-935, 1988.
8. Lyall, R. M.. Zilberstein, A., Gazit, A., Gilon, C., Levitzki. A., and Schles
singer, J. Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyro
sine kinase activity in living cells and EGF-stimulated cell proliferation. J.
Biol. Chem., 264: 14503-14509, 1989.
9. Yoneda, T., Nishikawa, N., Nishimura, R., Kato, I., and Sakuda. M. Three
cases of oral squamous cancer associated with leukocytosis, hypercalcemia,
or both. Oral Surg. Oral Med. Oral Pathol., 68: 604-611, 1989.
10. Yoneda, T., Aufdemorte, T. B., Nishimura, R., Nishikawa, N., Sakuda, M.,
Alsina, M., Chavez, J. B., and Mundy, G. R. Occurrence of hypercalcemia
and leukocytosis with cachexia in a human squamous cell carcinoma of the
maxilla in athymic nude mice. A novel experimental model of 3 concomitant
paraneoplastic syndromes. J. Clin. Oncol., 9: 468-477, 1991.
11. Honegger. A. M., Szapary, D., Schmidt, A., Lyall, R.. Van Obberghen, E.,
Dull, T. J., Ullrich, A., and Schlessinger, J. A mutant epidermal growth
factor receptor with defective protein tyrosine kinase is unable to stimulate
proto-oncogene expression and DNA synthesis. Mol. Cell. Biol., 7: 4568-
4571, 1987.
12. Honegger, A. M., Dull, T. J., Felder, S., Van Obberghen, E.. Bellot, F.,
Szapary, D., Schmidt, A., and Schlessinger, J. Point mutation at the ATP
binding site of EGF receptor abolishes tyrosine kinase activity and alters
cellular routing. Cell, 51: 199-209, 1987.
13. Aboud-Pirak, E., Hurwitz, E., Pirak, M. E., Bellot, F., Schlessinger, J., and
Sela, M. Efficacy of antibodies to epidermal growth factor receptor against
KB carcinoma in vitro and in nude mice. J. Nati. Cancer Inst., 80: 1605-
1611, 1988.
14. Laemmli, U. K. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.
15. Margolis, B., Zilberstein, A., Franks, C., Felder, S., Kremer, S., Ullrich, A.,
Rhee, S. G., Skorecki, K., and Schlessinger, J. Effect of phospholipase C-i
overexpression on PDGF-induced second messenger and mitogenesis. Sci
ence (Washington DC), 248:607-610, 1990.
16. Frindel, E. L., Malaise, E. P.. Alpen, E., and Tubiana, M. Kinetics of cell
proliferation of an experimental tumor. Cancer Res., 27; 1122-1131, 1967.
17. Yoneda, T., Alsina, M. M., Watatani, K., Bellot, F., Schlessinger, J., and
Mundy, G. R. Dependence of a human squamous cell carcinoma and asso
ciated paraneoplastic syndromes on the epidermal growth factor receptor
pathway in nude mice. Cancer Res., 52: 2438-2443, 1991.
18. Heimbrook, D. C., Stirdivant, S. M., Ahern, J. D., Balishin, N. L., Patrick,
D. R., Edwards, G. M., Defeo-Jones, D., FitzGerald, D. J., Pastan, L, and
Oliff, A. Transforming growth factor a-Pseudomonas exotoxin fusion protein
prolongs survival of nude mice bearing tumor xenografts. Proc. Nati. Acad.
Sci. USA, «7:4697-4701, 1990.
19. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato, G., and Mendelsohn,
J. Growth inhibition of human tumor cells in athymic mice by anti-epidermal
growth factor receptor monoclonal antibodies. Cancer Res.. 44: 1002-1007,
1984.
20. Dexter, D. L., and Leith, J. T. Tumor heterogeneity and drug resistance. J.
Clin. Oncol.. 4: 244-257. 1986.
21. Hurwitz, E., Burowski, D., Kashi, R., Hollander, N., and Haimovich, J. A
synergistic effect between anti-idiotype antibodies and anti-neoplastic drugs
in the therapy of a murine B-cell tumor. Int. J. Cancer, 37: 739-745, 1986.
22. Aboud-Pirak, E., Hurwitz, E., Bellot, F., Schlessinger, J., and Sela, M.
Inhibition of human tumor growth in nude mice by a conjugate of doxorubicin
with monoclonal antibodies to epidermal growth factor receptor. Proc. Nati.
Acad. Sci. USA, 86: 3778-3781, 1989.
23. Yoneda, T., Alsina, M. M., Chavez, J. B., Bonewald, L. F., Nishimura, R.,
and Mundy, G. R. Evidence that tumor necrosis factor plays a pathogenic
role in the paraneoplastic syndromes of cachexia. hypercalcemia, and leuko
cytosis in a human tumor in nude mice. J. Clin. Invest., 87: 977-985, 1991.
24. Davidson, N. E., Gelmann, E. P., Lippman. M. E., and Dickson, R. B.
Epidermal growth factor receptor gene expression in estrogen receptor-
positive and negative human breast cancer cell lines. Mol. Endocrinol., /:
216-223, 1987.
25. Reddy, K., Bradley, G., Yoneda. T., Mundy, G., and Osborne, C. K. Inhibition
of breast cancer cell growth in vitro by a receptor tyrosine kinase inhibitor
(tyrphostin). Proc. Am. Assoc. Cancer Res., 32: 92, 1991.
26. Glazer, R. I., Yu, G., and Knode, M. C. Analysis of tyrosine kinase activity
in cell extracts using nondenaturing polyacrylamide gel electrophoresis. Anal.
Biochem., 164: 214-220, 1987.
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Research.
on December 14, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

1991;51:4430-4435. Cancer Res
Toshiyuki Yoneda, Ray M. Lyall, Maria M. Alsina, et al.
and in Nude Mice in VitroTyrphostins on a Human Squamous Cell Carcinoma
The Antiproliferative Effects of Tyrosine Kinase Inhibitors
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