[CANCER RESEARCH 58. 5551-5558.
December I, 1998]
Retinole Acid and Interferon a Act Synergistically as Antiangiogenic and
Antitumor Agents against Human Head and Neck Squamous
Cell Carcinoma1
Mark W. Lingen,2 Peter J. Polverini, and NoëlP. Bouck
Departments of Pathology [M. W. L] and Microbiology-Immunology
[N. P. BJ and the R. H. Lurie Cancer Center, Northwestern University Medical and Dental Schools,
Chicago. Illinois 60611, and The Laboratory of Molecular Pathology, The University of Michigan School of Dentistry, Ann Arbor, Michigan 4HI09 /P. J. P. I
death (3). To improve long-term outcome, some form of chemopreventive treatment is essential.
Head and neck squamous cell carcinoma (HNSCC) is an aggressive
The use of chemopreventive agents in halting the development of
malignancy in which multiple independent lesions develop over time
squamous
cell carcinoma has been studied extensively in animal
throughout the mucosa of the upper aerodigestive tract. Therefore, the
models
of
oral
carcinogenesis and is under investigation in worldwide
comprehensive treatment of this neoplasm must include a chemoprevenclinical trials. The most successful agents used to date have been the
tive arm to hold premalignant lesions in check, a role well-suited to
retinoids (4-6). The biological mechanisms by which retinoids act to
antiangiogenic agents. Retinole acid (RA) and interferon a (IFN-a), drugs
with known biological activity against HNSCC when used individually,
prevent the development of new primary HNSCC lesions is unclear
are also inhibitors of angiogenesis. Here we show that they are remarkably
and controversial. In some tumor types, the effectiveness of retinoids
synergistic antiangiogenic agents able to inhibit both the growth and the
correlates with their ability to modulate growth of tumor cells them
neovascularization of HNSCC injected into the floor of the mouth of nude
selves by influencing proliferation, differentiation, and/or apoptosis
mice. The mechanism of action of these drugs as antiangiogenic agents was
(7-11). However, retinoids do not consistently inhibit the growth of
2-fold. They decreased the angiogenic activity of the tumor cells, and they
HNSCC tumor lines until doses rise several logs above therapeutically
caused the endothelial cells to become refractory to inducers of angiogen
achievable levels, suggesting that their effectiveness against this tu
esis. When tumor cells were treated in vitro with IFN-a A/D, there was a
mor type may also depend on additional activities of retinoids. Reti
dramatic drop in their secretion of interleukin-8, the major angiogenic
noids are also effective antiangiogenic agents (12-15), and recent
factor produced by these tumors. When combined with RA, which causes
tumor cells to secrete an inhibitor of angiogenesis, there was a synergistic
work suggests that the chemopreventive activities of RA against the
inhibition of both tumor cell growth and secreted angiogenic activity. The
development of HNSCC may be due at least in part to its ability to
combination of RA and IFN-a also acted synergistically on endothelial
inhibit tumor-induced angiogenesis via two distinct mechanisms (16,
cells by reducing their responsiveness to both interleukin-8 and tumor
17). At clinically relevant concentrations, it makes endothelial cells
conditioned media. Doses of each drug could be reduced by two logs
less responsive to angiogenic factors and causes HNSCC cells to
without loss of activity. When animals bearing human HNSCC tumor cells
become antiangiogenic by secreting a protein that can inhibit neovas
were treated systemically with a combination of RA and IFN-a A/D at
cularization.
doses that were ineffective when used alone, dramatic decreases in both
Although its efficacy has been demonstrated in clinical trials (4-6),
tumor growth and tumor angiogenesis were seen. These data suggest that
the widespread use of RA as a chemopreventive agent has been
the use of antiangiogenic mixtures may be a particularly effective way to
design future chemoprevention protocols against HNSCC.
hampered by the toxic side effects experienced by individuals who are
taking the drug for prolonged periods of time (5). Therefore, there is
an intense search for other chemopreventive agents that may act
synergistically with RA to reduce the toxicity and improve long-term
INTRODUCTION
HNSCC3 is an aggressive epithelial malignancy that is now the patient compliance without sacrificing efficacy and clinical outcome.
One possibility are the IFNs.
sixth most common neoplasm in the world today. At present projec
IFNs are multifaceted agents long known for their ability to influ
tions, —50,000cases in the United States and —500,000cases world
ence proliferation, differentiation, and the immune system (18). In
wide will be diagnosed in 1997 (1). Despite numerous advances in
addition, IFNs also possess antiangiogenic activity. IFNs can inhibit
treatment using the most recent protocols for surgery, radiation, and endothelial cell proliferation and migration in vitro (19-23) as well as
chemotherapy, the long-term survival has remained —50%for the last
corneal neovascularization and embryonic neovascularization in vivo
30 years (2). This poor prognosis is due in part to the frequent
development of multiple additional primary tumors. The occurrence (24). They can also decrease the amount of the angiogenic factor basic
of multiple primary tumors can be particularly devastating for those fibroblast growth factor that is secreted by renal cell carcinoma tumor
whose initial lesions are small. Although their 5-year survival rate cells (25). IFN-a has also been used effectively as an antiangiogenic
after the first primary tumor is considerably better than 50%, second agent to treat juvenile hemangiomas (26).
Most patients receiving high doses of IFN experience some degree
primary tumors are their most common cause of treatment failure and
of acute toxicity as well. The most common side effects are flu-like
symptoms that include chills, fever, myalgia, and headache. Chronic
Received 6/10/98; accepted 10/2/98.
exposure results in additional symptoms including fatigue, anorexia,
The costs of publication of this article were defrayed in pan by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
weight loss, dizziness, and some hematological toxicity (27). Much
18 U.S.C. Section 1734 solely to indicate this fact.
like RA, its side effects make it unlikely to be acceptable as a
' This work was supported in part by the N1H grants DE00313 (to M. W. L.). CA52750
and CA64239 (to N. P. B.), HL39926 and CA64416 (to P. J. P.). and CA60553 (to R. H.
chemopreventive agent used chronically at current dose regimens.
Lurie Cancer Center).
However, the long-term use of both RA and IFN-a at lower doses
2 To whom requests for reprints should be addressed, at Loyola University Medical
might hold some promise if they were found to possess synergistic
Center, Department of Pathology. Cardinal Bernardin Cancer Center. 2160 South First
Avenue. Maywood. IL 60153. Phone: (708) 327-3141: Fax: (708) 327-3342; Email:
activity. The fact that they bind to distinct receptors and exert their
MLingen @Luc.edu.
effects via different signaling mechanisms suggest that their chemo
3 The abbreviations used are: HNSCC. head and neck squamous cell carcinoma: RA.
retinoic acid; CM, conditioned media.
preventive activities could be more than additive.
5551
ABSTRACT
Downloaded from cancerres.aacrjournals.org on July 12, 2017. © 1998 American Association for Cancer Research.
RKTlNOmS.
INÕÕ-RM-RONS.ANl¡I(K¡!-.NE-:.SIS.
AND ORAL CANCER
B
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IFN-alpha (lU/ml)
IFN-alpha (lU/ml)
Fig. I. Effects of IFN-a on (he proliferation and angiogenic activity of HNSCC cells. In A. the cell lines SCC-4. SCC-9. and the SCC-25 were treated with the indicated
concentrations of IFN-a A/D for 7 days. CM were collected during the final 24 h and assayed for their ability to stimulate endolhelial cell migration. Values are reported as a percentage
of maximal migration; bars, SE. In B. separate experiments, human tumor cells were assayed for their ability to proliferate in the presence of increasing concentrations of IFN-a. Values
are reported as the corrected absorbance at 492 nm.
A variety of data supports the possibility that the combination of
retinoids and IFNs may have additive or synergistic antitumor effects
on certain neoplasms. RA and IFN have been found to be synergistic
in their abilities to inhibit the proliferation of a number of different
types of tumor cells including malignant melanomas, murine embyronal, breast, ovarian, and some squamous cell carcinoma cell lines
(28-34). Phase I and II clinical trials using both RA and IFN to treat
advanced solid tumors have reported some promising results (28, 35),
with variable levels of clinical response for non-small cell carcinoma
of the lung, advanced renal cell carcinoma, metastatic melanoma,
gastrointestinal adenocarcinoma, endometrial carcinoma, and treat
ment of advanced stages of squamous cell carcinoma of the skin,
esophagus, cervix, vulva, penis, and head and neck.
This report looks toward the long-term use of RA and IFN-a
together in a chemopreventive setting that would depend primarily on
their antiangiogenic activities. It is shown that both agents act on
HNSCC tumor cells to limit their ability to stimulate angiogenesis as
well as on microvascular endothelial cells to prevent their response to
angiogenic factors. Dramatic synergism was seen between these
agents both against tumor cells and the normal endothelial cells in
vitro. In vivo, they reduced angiogenesis and growth of HNSCC tumor
cells, suggesting that their use in combination could be of significant
benefit in the long-term chemoprevention of these aggressive neo
plasms.
MATERIALS
Bovine adrenal microvascular endothelial cells, BP10T8 (a kind gift of
Judah Folkman. Harvard University. Boston, MA), were grown in DMEM
with 10% donor calf serum, 100 jig/ml endothelial cell mitogen (Biomedicai
Technologies Inc.. Stoughton, MA), 200 mM glutamine, 100 units/ml penicil
lin, and 50 fig/ml streptomycin. Endothelial cells were cultured at 37°Cin a
8% CO2-92% air environment in humidified incubators.
For treatment of cells with RA, stocks of 10"' M all-/ram-RA
The desired dilutions were made directly into the culture medium, keeping the
final concentration of DMSO at <0.1%. Cells were treated with RA for the
times and concentrations indicated for each individual experiment and refed
with fresh media containing RA every third day during treatment. Control
cultures were treated in parallel with culture media containing 0.1 % DMSO
alone. For treatment of cells with IFN-a A/D, stock solutions of recombinant
human interferon-a A/D (BioSource International, Camarillo, CA) were pre-
Table 1 Corneal neovasculari~Ã-ition in response lo CM from drug-treated
source"ControlDMEMIL-8Tumor
Media
in humidified incubators.
Tested
cells0/3
alone
+ CM from
untreated tumor
(0)2/2(100)2/2(100)0/3
CMSCC-4UntreatedIFN
Cell Culture. Human squamous cell carcinoma cell lines SCC-4, SCC-9,
SCC-15. and SCC-25 were purchased from the American Type Culture Col
lection. These cells were grown in DMEM Ham's F-12 (1:1) supplemented
in a 5% CO2-95% air environment
tumor cells
(%)Tested
of implants
AND METHODS
with 10* fetal bovine serum, 0.4 /xg/ml hydrocortisone. 100 units/ml penicil
lin, and 50 ¿ig/mlstreptomycin. HaCat (36), an immortal human keratinocyte
cell line (kindly provided by Brian Nickoloff. University of Michigan), was
grown in DMEM supplemented with 10* fetal bovine serum. 100 units/ml
penicillin, and 50 ¿tg/mlstreptomycin. All keratinocytes were cultured at 37°C
(Sigma
Chemical Co., St. Louis. MO) were prepared in DMSO and stored at —
20°C.
treatedIFN
treatedSCC-9UntreatedIFN
4- RA
(0)0/2
(0)4/4(100)0/3
(75)1/3
(33)ND3/3(100)0/3
treatedIFN
treatedSCC-25UntreatedIFN
+ RA
(0)0/3
(0)3/3(100)0/3
(0)ND2/2(100)0/2
treatedIFN
(0)0/3
+ RA treatedPositivecomeas/No,(0)ND'1NDND3/4
(0)
" Where indicated, cells were treated with 1000 IU of IFN-a for 7 days, and CM were
collected during the final 24 h. Media were incorporated into pellets and implanted into
rat corneas, and neovascularization was assessed 7 days later.
h ND. not done.
5552
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RKTINOIDS.
INTERFERONS.
ANOIOGHNliSIS.
of maximal migration to a positive control. Each substance was tested in
quadruplicate in each experiment, and all experiments were repeated at least
twice.
Corneal Neovascularization Assay. In vivo angiogenic activity was as
sayed in the avascular cornea of F344 female rats (HarÃ-anLabs. Madison. WI)
as described previously (16). Test substances were incorporated into sterile
Hydron (Interferon Sciences. New Brunswick, NJ) pellets inserted into a
surgically created corneal pocket within 1.5 mm of the limbus. Corneas were
observed every other day until day 7. when the animals were anesthetized and
perfused with PBS. followed by colloidal carbon to stain the vessels. Re
sponses were scored as positive when vigorous, sustained, directional in
growth of capillary sprouts and hairpin loops toward the implant were detected.
Negative responses were recorded when no growth was detected or when there
was only an occasional sprout or hairpin loop with no evidence of sustained
growth. Negative controls consisted of Hydron pellets containing DMEM/F- 12
alone. Media were incorporated into pellets at a total concentration of l /ig of
total protein/cornea.
The in vivo effects of systemic RA and IFN-a on neovascularization
[771 Untreated Tumor Cells (1)
I
I IFN Treated Tumor Cells (2)
PP
1 +2
L\N~ Retinole
AcidTreatedTumorCells(3)
1 +3
60
tr
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SCC-4
induced by tumor CM were tested by placing Hydron pellets containing tumor
CM into the cornea and then injecting the animals i.p. daily with DMSO
vehicle. all-ira«.v-RAalone. IFN-a alone, or RA and IFN-a for 7 consecutive
BSA
days. The rats were subsequently anesthetized and perfused, and the corneas
were scored for neovascular responses. Toxicity of drug treatment was mon
itored by weighing the animals daily. No animal experienced a weight loss of
>10% during the treatment regimen (data not shown).
Cell Proliferation Assay. The in vitro proliferation of endothelial cells was
determined using the CellTiter 96 AQueous Non-Radioactive Cell Prolifera
tion Assay kit (Promega Corp.. Madison, WI). Cells were plated at a density
of 1000 cells/well in a gelatini/.ed 96-well plate in appropriate media. After
\
\ KXX
\
\
\
\
\
\
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\È5//;/ñi\\\\TyK
X
SCC-9
Tumor Conditioned
SCC-25
Media
Fig. 2. CM from tumor cells treated with IFN-a does not contain angioinhibilory
activity. CM from untreated and IFN-a A/D-trealed or RA-treated cells were assayed
alone or mixed together ina 1:1 ratio to determine their ability to stimulate or inhibit
endothelial cell migration. Values are reported as the number of cells migrated per 10 high
power fields; bars, SE.
pared by dilution in PBS and stored at —20°C.IFN-a A/D is a recombinant
human IFN-a that is highly reactive on human, mouse, rat, bovine, primate,
canine, equine, ovine, feline, and hamster cells (37). Desired dilutions were
made directly into the culture medium. Cells were treated with IFN for the
times and concentrations indicated for each individual experiment and refed
with fresh media containing IFN every third day. Control cultures were treated
and refed with culture medium containing PBS alone.
Viability of the drug-treated tumor cells was determined by plating in
triplicate at 200 cells/plate
and counting colonies after
was decreased as well as
concentrations greater than
ANI) ORAI, CANCER
in media containing varying drug concentrations
6 days. At high doses in RA, the size of the clones
their number, but this effect was only seen at
10 5 M (data not shown). Viability of endothelial
8 h, the cells were starved overnight in DMEM containing 0.1% BSA. then
refed with their appropriate media containing the indicated concentrations of
test substances. The cells were refed again on day 3. and the assay was
performed on day 6. Results are expressed as corrected absorbance, which
represents the actual absorbance of each well, as recorded at 490 nm. minus the
actual absorbance of 1000 cells determined on day 0.
The proliferation of tumor cells was determined in a similar fashion, except
tumor cells were plated at a density of 500 cells/well and starved overnight.
Results are expressed as the corrected absorbance, which represents the actual
absorbance of each well, as recorded at 490 nm. minus the actual absorbance
of 500 cells determined on day 0.
Quantification of IL-8 in CM. The amount of IL-8 present in the CM of
the untreated and treated tumor cells was determined using a Quantikine
Immunoassay kit (R&D Systems. Minneapolis. MN). Briefly, l /ig of total
protein from each CM sample was assayed in duplicate for the presence of IL-8
using the sandwich ELISA technique. The average of the two assays is
presented. Individual assays of the same sample varied from one another by
Assay for in Vivo Tumor Growth. The ability of RA and IFN to inhibit m
vivo angiogenesis and tumor growth was tested as described previously (38),
cells was monitored by trypan blue exclusion. A significant decrease in cell
viability was only observed at concentrations greater than 10~6 M RA or
10,000 lU/ml of IFN-a A/D (data not shown).
Serum-free tumor CM was generated as described previously (17). Add-
Table 2 Secretion of IL-K hy iunior cells treated with RA and IFN-a
Media
sourceNHOKHaCatSCC-4SCC-9SCC-
IL-8/ngprotein75902520015050800750200160013
total
back experiments were performed to ensure that the concentration and dialysis
of CM from the RA-. IFN-a, and the RA/IFN-a-treated cells resulted in the
complete removal of the drugs (data not shown).
Endothelial Cell Migration Assay. The endothelial cell migration assay
was performed as described previously (16). Briefly. BP10T8 cells were
starved overnight in DMEM containing 0.1% BSA. harvested, resuspended
into DMEM with 0.1% BSA, plated on the bottom side of a modified Boyden
chamber (Nucleopore Corporation, MD). and allowed to attach in an inverted
chamber for 2 h at 37°C.The chamber was then re-inverted: test substances ( 1
fig of total protein per test compound in a volume of 50 /j.1) were added to the
wells of the upper chamber, and cells were allowed to migrate for 4 h at 37°C.
Membranes were recovered, fixed, and stained, and the numbers of cells that
had migrated to the upper chamber per 10 high power fields were counted.
Background migration to DMEM + 0.1% BSA was subtracted, and the data
were reponed as the number of cells migrated per 10 high power fields ( X400)
or, when results from multiple experiments were combined, as the percentage
15SCC-25Treatment"Noneall-/r«n.v-RAIFN-aNoneall-;r«n.s-RAIFN-aNoneall-rrüMÃ--RAIFN-aNon
' Tumor cells treated with 10
M RA or 1000 lU/ml for 7 days.
5553
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RETINOIDS. INTERFERONS, ANÕiKXil.NI.SIS. AND ORAL CANCER
B
IFN alpha (lU/ml)
IFN alpha (ID/ml)
10
—I—
D
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100
1000
10
10000
100
1000
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100
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Retinoic Acid [M]
Retinole Acid [M]
IFN alpha Alone
IFN alpha+10'8 RA
IFN alpha+ 10'7 RA
IFN alpha+ 10'6 RA
IFN alpha
IFNalpha + 10'8RA
IFN alpha+ 10"7RA
IFN alpha+10"6 RA
RA Alone
10'6RA
Fig. 3. Syncrgislic effects of RA and IFN-a on the inhibition of angiogenic activity and proliferation of human HNSCC cells. In A, tumor cell lines were treated with increasing
concentrations of cither all-r/'iin.v-RA, IFN-a A/D, or with increasing doses of IFN-a A/D and IO "KM RA, or 10~7 M RA. or 10 h M RA for 7 days. CM were then assayed for their
ability to stimulate endothelial cell migration. Values arc reported as a percentage of maximal migration: bars, SE. B, tumor cells assayed for their ability to proliferate when treated
with increasing concentrations of either RA alone. IFN-or alone, or with increasing doses of IFN-a A/D and 10"* M RA. 10" 7 M RA. or 10~6 M RA for 7 days. Values are reported
as the corrected absorhancc al 490 nm.
with some minor modifications.
Briefly, a 50-/xl bolus of 6 X 10 6 SCC-9
tumor cells were injected into the floor of the mouth of athymic nude mice via
a 26-gauge needle. Following introduction of the tumor cells, the mice were
given daily i.p. injections of RA alone (0.5 mg/kg/day or 1 mg/kg/day). IFN-a
alone ( 1000 IU/kg/day or 5(XX)IU/kg/day), or RA (0.5 mg/kg/day) in combi
nation with IFN-a (KXX) IU/kg/day). Twenty-one days after implantation of
50
the tumor cells, the mice were sacrificed, and the lesions were carefully
excised to include only tumor tissue. The tumor masses were immediately
measured with calipers and weighed. The mice were weighed on a daily basis
as a means of measuring relative toxicities of treatment. The RA. IFN-a. and
the RA/IFN-a treatments did not result in a >10% decrease in weight of the
I• 40
ii
SCC-9
30
mice after 21 days of treatment (data not shown).
Determination of Microvessel Densities. Formalin-fixed, paraffin-embed
ded tissue from each tumor was generated to quantify differences in vessel
densities in the tumors of the treated and untreated animals. Serial sections of
5 /im in thickness were prepared and processed for standard avidin-biotiny-
20
BSA
•s
lated en/.yme complex immunohistochemistry.
Antigen epitopes were un
masked by microwaving the specimens in \% /ine sulfate for 3 min. The
biotin-conjugated rat anti-mouse CD-31 (PECAM-1) antibody (PharMingen.
Los Angeles. CA), which recognizes the M, 130.000 integral membrane
protein used on all sections. Microvessel density was assessed using the
technique described by Vermeulen el al. (39). The region containing the most
intense area of tumor neovascularization was chosen for counting in each of
the tumors. Individual microvessels were counted using a X200 field (X20
objective lens and x 10 ocular lens). Any brown staining endothelial cells that
were clearly separate in appearance were counted as individual vessels. Results
were expressed as the highest number of microvessels observed at X200 in the
"hot spot" region of each individual tumor.
10
Õ
SCC-9 RA
SCC-9 IFN
SCC-9 RA/IFN
Tumor Cell Conditioned Media
Fig. 4. SCC-9 cells treated with both RA and IFN-a stilt secrete angioinhibitory
activity. CM were collected from tumor cells treated with either 10 ' * VIRA or 10 ~* M RA
plus 10 IU IFN-a for 7 days, concentrated, and assayed for their ability to stimulate
endothelial cell migration. Values are reported as the number of cells migrated per 10 high
power fields: bars. SE.
5554
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RETINOIDS.
INTERFERONS.
ANGIOCENESIS.
AND ORAL CANCER
B
IFN alpha (IU/ml)
IFN alpha (lU/ml)
10
3
'xE
100
10
1000
1000
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100
100
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Retinole Acid [M]
Retinole Acid [M]
IFN alpha Alone
IFN alpha+ 10'8 RA
IFNalpha + 10'7RA
IFN alpha+ 10'6 RA
IFN alpha
IFNalpha+10'8RA
IFN alpha+10'7 RA
IFN alpha+10'6 RA
10'6RA
RA Alone
Fig. 5. Retinole acid and IFN-a have a synergistic effect on the inhibition of endothelial cell migration and proliferation. In A. capillary cndothclial cells (BP10TS) were assayed
for their ability to migrate toward 1L-8 in the presence of increasing doses of either RA. IFN-a. or with increasing amounts of IFN-«A/D and IO"* M RA, or IO"7 M RA. or IO'6
M RA. Values for each set of experiments are reported as the percentage of maximal migration to IL-8 after subtraction of background migration to BSA: bars. SE. In ß.in separate
experiments, capillary endothelial cells (BP10T8) were assayed for their ability to proliferate in the presence of increasing doses of either RA. IFN-a. or with increasing amounts of
IFN-a A/D and 10 * M RA, IO"7 M RA, or IO"*1M RA. Values are reported as the corrected absorbance at 492 nm.
RESULTS
Response of HNSCC Tumor Cells to RA and IFN-a. HNSCC
cell lines treated with increasing doses of IFN-a A/D lost their ability
to induce angiogenesis in a dose-dependent manner. Cells were grown
in various concentrations of IFN-a for 7 days, and their secretions
during the last 24 h were collected as serum-free CM. The ability of
these media to induce endothelial cell migration was reduced to
background levels when the cells had been treated with as little as
1000 IU/ml, with an ED50 of 100 IU/ml (Fig. \A). Their media were
also unable to induce neovascularization in vivo (Table 1). In contrast
to their marked inhibition of the secretion of angiogenic activity, a
concentration of 100 IU/ml IFN-a A/D had only modest inhibitory
effects on the proliferation of the tumor cells in vitro (Fig. Iß).
We have demonstrated previously that RA treatment causes
HNSCC cells to become antiangiogenic as a result of the secretion of
a RA-inducible inhibitor of angiogenesis ( 17). However, unlike RAtreated tumor cells, the fall in the angiogenic activity seen in IFN-atreated cells was not due to a gain in inhibitory activity. Their CM
were unable to block angiogenesis induced by CM from untreated
tumor cells when tested in vitro (Fig. 2) or in vivo (Table 1). Rather,
the IFN-induced loss of angiogenic activity was due to a significant
decrease in inducing activity. The levels of secreted IL-8, the primary
angiogenic factor secreted by these cells (17), plummeted after IFN
treatment (Table 2).
Because both RA and IFN-a inhibited angiogenesis induced by
HNSCC via different mechanisms, we tested the ability of these
compounds to act in a synergistic fashion. When tumor cells were
treated with both RA and IFN-a, a synergy was seen between the two
agents in their ability to inhibit tumor cell-induced angiogenesis
(Fig. 3A). Media collected from tumor cells treated with as little as
10~8 M RA, and 10 IU/ml of IFN-a also demonstrated a markedly
decreased ability to induce endothelial migration, reproducing effects
that required either 10~6 RA or 1000 IU/ml of IFN-a if used alone
(Fig. 3A). CM from tumor cells treated with both RA and IFN-a
continued to contain inhibitory activity elicited by RA treatment
because the CM was able to block the induction of angiogenesis by
CM from untreated tumor cells in vitro (Fig. 4) and in vivo (Table 1).
These drugs also had synergistic effects on tumor cell proliferation.
Tumor cells treated with as little as 10~K MRA and a little as 10 IU/ml
of IFN-a demonstrated a reduction in proliferation (Fig. 3S). This
level of inhibition was similar to that seen when tumor cells were
treated with either nonphysiological doses of RA or with as much as
10,000 IU/ml of IFN-a alone.
Response of Capillary Endothelial Cells to RA and IFN-a.
Unlike RA (16), the migration of endothelial cells was not inhibited
immediately by the addition of IFN-a to migrating cells (data not
shown). A 24-h pretreatment of the endothelial cells was necessary to
see an inhibition of their ability to migrate, which was effectively
eliminated at a dose of 100 IU/ml (Fig. 5A). In addition, the prolif
eration of capillary endothelial cells was inhibited by IFN-a in a
5555
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RETINOIDS. INTERFERONS, ANGKXiHNIiSIS.
Table 3 Svsiemic IFN-a A/D inhibits corneal neovascularizalion induced by IL-8
AND ORAL CANCER
slow the growth of human SCC tumor cells in nude mice. SCC-9 cells
were injected into the floor of the mouth of nude mice, and these mice
corneas/
Substance
implantedControlsDMEMIL-8IL-8
treatedNoneNonel,OOOIU/kg/day2.500IU/kg/day5.000IU/kg/day1
No. of(%)0/2(0)2/2(100)2/2(100)2/2(100)0/2
implants
were then treated with daily doses of either RA (0.5 mg/kg/day or 1
IFN-a"IL-8IL-8IL-8IL-8Animals
+
(0)0/2
0.0001U/kg/dayPositive
(0)
" Animals whose corneas had been implanted with the indicated substances were
treated with various doses of IFN-a A/D every day for 7 days, and corneal neovasculari/alion was assessed.
Table 4 SvxtemicIFN-a reduced corneal neovasculari-ation induced b\ human tumor
CM
Positive comeas/No, of implants (%)
source"ControlsDMBMIL-8Tumor
Media
alone0/3
+ 5000 IU
(A/D)/kg/day0/2
IFN-a
(0)3/3(100)2/2(100)3/3(100)2/2(100)Tested
(0)0/3
(0)0/2
CMSCC-4SCC-9SCC-25Tested
(0)0/3
(0)0/2
(0)
" The indicated media were implanted into the corneas of rats and treated with systemic
doses of IFN-a A/D, and corneal neovasculari/.ation was assessed after 7 days.
dose-dependent manner with an EDS()of approximately 50 lU/ml (Fig.
5ß).Similar results for the inhibition of endothelial cell migration and
proliferation by IFNs have been observed by others as well ( 19-23).
The combination of RA and IFN-a had a marked effect on both the
migration and proliferation of endothelial cells. Cells treated with as
little as 10"x M RA and 10 ILJ/ml of IFN-a demonstrated the same
level of inhibition of migration as cells treated with 10~fi MRA or 100
ILJ/ml of IFN-a alone (Fig. 5A). Similarly, cells treated with as little
as 10"s M RA and 10 lU/ml of IFN-a demonstrated the same level of
inhibition of proliferation as cells treated with either nonphysiological
doses of RA or 100 IU of IFN-a alone (Fig. 5B).
Synergism Between RA and IFN-a after Systemic Administra
tion. To confirm that the antiangiogenic effects of IFN-a on endo
thelial cells seen in vitro truly reflected their effect on these cells in
vivo, Hydron pellets containing either IL-8 or HNSCC CM were
placed into rat corneas. These rats were then treated with various
systemic doses of IFN-a for 7 days and subsequently evaluated for the
presence of corneal neovascularization. Vigorous sustained vessel
ingrowth was observed in animals that were treated with carrier alone.
However, there was considerable inhibition of vessel ingrowth in the
animals treated with doses of 5000 IU/kg/day or greater, demonstrat
ing that IFN-a A/D can inhibit in vivo neovascularization (Tables 3
and 4).
We have demonstrated previously similar inhibition of in vivo
neovascularization by systemic RA (16). To determine whether lower
doses of each drug could act synergistically to inhibit in vivo neovas
culari/.ation, rats were treated with the combination of RA and IFN-a
A/D. Hydron pellets containing tumor CM were placed into the rat
cornea, and the animals were subsequently treated for 7 days with
both RA and IFN-a via i.p. injections. Rats treated with as little as 0.5
mg/kg/day RA and KXK)IU/kg/day of IFN-a A/D demonstrated a
marked decrease in corneal neovascularization. These doses were as
effective as either I mg/kg/day of RA or 5000 IU/kg/day of IFN-a
alone (Table 5).
To determine whether these synergies extended to real tumor angiogenesis and tumor growth, we tested the ability of RA and IFN to
mg/kg/day), IFN-a (1000 IU/kg/day or 5000 IU/kg/day), or both RA
(0.5 mg/kg/day) and IFN-a (1000 IU/kg/day). After 21 days of
treatment, control tumors weighed 220 ±30 mg. When used individ
ually, the higher doses of both RA (1 mg/kg/day) and IFN-a (5000
IU/kg/day) caused a reduction in tumor weight when compared with
controls. Tumors from RA-treated animals weighed 73.3 ±5.7 mg,
whereas the tumors from the IFN-a-treated
animals weighed
76.7 ±5.7 mg. No decrease in tumor weight was observed in the
animals treated individually with the lower doses of either RA (0.5
mg/kg/day) or IFN-a (1000 IU/kg/day) when compared with control
animals (Table 6). However, the tumors from the animals treated
concurrently with RA (0.5 mg/kg/day) and IFN-a (1000 IU/kg/day)
weighed only 16.7 ±5.7 mg (Table 6). These results demonstrate that
the combination of RA and IFN-a can act dramatically in a synergistic
fashion to inhibit HNSCC tumor cell growth.
The ability of RA and IFN-a to inhibit tumor growth was depend
ent on the continuous administration of the drugs. When SCC-9 cells
were injected into the floor of the mouth of nude mice, the mice were
treated with RA (0.5 mg/kg/day) and IFN-a (1000 IU/kg/day) for 21
days; the drug treatment was then discontinued, and the tumors began
to grow. After 21 days of RA/IFN-a treatment, the mice had small
palpable tumors that grew rapidly to an average weight of 150 ±17
mg during the 14 days after removal of the drug (Table 6).
The observed inhibition of tumor growth in drug-treated animals
was due at least in part to the antiangiogenic activity of RA and
IFN-a. Animals treated individually with the higher doses of RA ( 1
mg/kg/day) or IFN-a had markedly decreased microvessel densities
when compared with controls. The mean microvessel densities of the
control tumors was 104 ±23, whereas the mean densities for the
RA-treated and the IFN-a-treated animals were 47 ±17 and 55 ±18,
respectively. Animals treated with the lower doses of either single
agent had vessels densities that were similar to controls (Table 6).
Table 5 SystemicRA anil IFN-a act synergistically in vivo to inhibit the induction of
cornent nemasculari-dtitm
Positive corneas/No, of implants (%)
Media source
Tested alone
Tested + 1000 IU IFN-a
(A/DI/kg/day + indicated
amounts of all-irans-RA
ControlDMEMSCC-9Tumor
(0)2/2(100)NDNDNDND"ND0/3
CMSCC-9
mg/kg/daySCC-9
+ 1
mg/kg/daySCC-9
+ 0.5
+ 0.25 mg/kg/day0/3
' ND. not done.
(0)0/3
(0)3/3(100)
Table 6 RA and IFN-a synergistically inhibit both tumor growth ami
microvessel density
TreatmentControlRA0.5
weights
(mg)220
density"104
30210
±
±2399
mg/kg/day1
mg/kg/dayIFN-a1000
±4573.3
6213±
±2147
1791
±
IU/kg/day5000
IU/kg/dayRA/IFN-a0.5
±4176.7
716.7±
1555
±
1821
±
IU/kg/dayRA/IFN-a
mg/1000
6150±
±988
(21 days) and no drug ( 14 days)Tumor
±17Microvessel
±12
" The mean of individual x200 fields counted from each tumor (n = 3 for each dosage
tested), where the greatest amounts of neovascularization were observed.
5556
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. IMT:RH:RONS.
ANGIOGENI-SIS.
ANDORALCANCER
However, the mean microvessel density of the RA/IFN-a-treated
animals was markedly reduced, with an average of 21 ±9 vessels per
hot spot. In addition, the tumors that were initially treated with RA
and IFN-a for 21 days and then remained drug free for 14 days
regained a microvessel density similar to that of the control tumors
(Table 6).
DISCUSSION
Squamous cell carcinoma is an aggressive malignancy that often
develops as multiple independent lesions throughout the mucosa of
the upper aerodigestive tract. Therefore, the comprehensive treatment
of this disease must address not only the initial primary neoplasm but
also prevent the progression of the premalignant lesions lurking
throughout the rest of the mucosal surfaces. The need to treat these
premalignant lesions has resulted in a search for chemopreventive
agents that can halt or even reverse their malignant progression.
Chemopreventive agents that act to inhibit angiogenesis may provide
a very powerful way to limit the growth of these premalignant lesions,
for if they are prevented from inducing new blood vessels, their
dormant phase of growth should be greatly extended. Here we show
that RA and IFN-a, drugs known to possess biological activity against
HNSCC, act in part by synergistically inhibiting tumor-induced an
giogenesis and thus may be most appropriate in a chemopreventive
setting.
Used alone, IFN-a caused HNSCC tumor cells to switch from
being angiogenic to nonangiogenic. However, unlike the switch in
duced by RA, the alteration of the angiogenic phenotype induced by
IFN-a was not due to the induction of the secretion of an inhibitor of
angiogenesis. Rather, IFN treatment caused a marked decrease in the
secretion of IL-8, the major inducer of angiogenesis secreted by
HNSCC tumor cells (17). This is consistent with previous observa
tions that IFN can down-regulate the production of IL-8 in fibroblasts
(40, 41). A number of other agents have been shown to inhibit the
transcription and translation of IL-8 message and protein in different
cells including glucocorticoids (42), 1,25-dihydroxycholecalciferol
(43), IL-4 (44), and dexamethasone (45). However, the inhibition of
IL-8 gene expression continues to be a poorly understood phenome
non that appears to be both cell type and agent specific. IFN can also
down-regulate the secretion of another angiogenic factor, basic fibroblast growth factor, in renal cell carcinoma tumor cells (24), suggest
ing that the direct antiangiogenic activity of IFN-a on tumor cells may
affect the ability of a spectrum of tumors to secrete a variety of
angiogenic molecules.
As expected from their distinct mechanisms of action, the combi
nation of RA and IFN-a demonstrated marked synergistic activity. A
decrease in angiogenic activity was observed in tumor cells that were
treated with concentrations of RA and IFN-a that were 2 logs below
those needed individually. Such a decrease in the amount of drug
should dramatically decrease the toxic side effects, particularly be
cause each drug elicits its own unique and nonoverlapping types of
symptoms. These findings are in keeping with the ability of IFN to act
in a synergistic or additive antiangiogenic fashion with AGM-1470
and minocycline, both potent inhibitors of angiogenesis (46, 47).
The synergistic effects of RA and IFN-a on endothelial cells were
equally impressive. Endothelial cell migration and proliferation were
strongly inhibited by significantly smaller doses of both drugs when
used together. The combination of the two drugs allowed for a one and
one-half log decrease in the concentration of RA and a 2 log decrease
in IFN-a. The same synergistic antiangiogenic effects were also
observed in vivo when systemic doses of RA/IFN strongly inhibited
corneal neovascularization. Initial experiments demonstrated that the
dosage of RA could be cut in half and the amount of IFN-a reduced
5-fold to achieve complete inhibition of corneal neovasculari/.ation.
Because the overall toxic side effects from RA are more severe than
those observed with IFN treatment, additional in vivo experiments are
planned to determine whether the dosage of RA can be decreased even
lower while the levels of IFN remain at somewhat higher doses.
The inhibition of tumor cell proliferation was an additional benefit
of combined RA/IFN-a treatment. Neither agent alone was particu
larly effective at inhibiting tumor cell proliferation until these cells
were treated with extremely high doses of drug. However, when
combined, there was a strong inhibition of proliferation at the same
relative doses where the synergistic effects on the induction of angio
genesis were observed. Synergistic antiproliferative activities of RA
and IFN-a on a number of different types of tumor cells has also been
observed by others in vitro assays (28-34).
The in vivo ability of systemic RA and IFN-a to slow the growth of
HNSCC tumor cells at doses where neither was effective alone is
particularly intriguing. This treatment seemed to hold the growth of
the tumor nests to a size where the requirement for angiogenesis is
minimal for continued survival (48, 49). These in vivo effects may be
due in part to the direct inhibition of tumor cell growth. However, the
ability of RA and IFN-a to block in vivo corneal neovasculari/.ation as
well as reduce tumor vessel densities in vivo strongly supports the fact
that the inhibition of angiogenesis may play a key role in the activity
of these two compounds.
These results are in keeping with recent experimental evidence that
has demonstrated that growth of small nests of cells can be stunted by
a variety of agents that have the common ability to inhibit angiogen
esis (50-55). Therefore, these results imply that the combination of
RA and IFN-a may be capable of holding small premalignant, ma
lignant, as well as micrometastatic lesions in check by inhibiting their
ability to stimulate angiogenesis.
The long-term goals of chemoprevention must be 2-fold. Treatment
protocols must be developed that can be easily taken by individuals
who have had a previous malignancy as well as by individuals who
are at high risk for their initial squamous cell carcinoma. The toxic
side affects must be extremely low to achieve widespread and longterm compliance. This would be particularly important in the case of
high-risk patients, with microscopically verified dysplastic lesions,
who have yet to have their first malignancy, and the treatment must be
capable of halting the growth of premalignant lesions before they
emerge clinically. The data presented in this report suggest that one
possible therapeutic approach may be to treat patients with a mixture
of synergistic antiangiogenic agents. RA and IFN-a appear to be
especially good candidates for this type of therapy. If such an anti
angiogenic chemoprevention protocol could be developed, it may
provide us with the ability to prevent the development of HNSCC and
perhaps other malignancies as well.
REFERENCES
1. Vokes. E. E.. Weichselbaum. R. R.. Lippman. S. M., and Hong. W. K. Head and neck
cancer. N. Engl. J. Med., 32K: 184-194. 1993.
2. Boring. C. C.. Squire. T. S.. Tong. T.. and Montgomery, S. Cancer statistics. CA.
Cancer J. Clin.. 44: 7-26. 1994.
3. Lippman. S. M.. and Hong. W. K. Second malignant tumors in head and neck
squamous cell carcinoma: the overshadowing threat for patients with early-stage
disease. Int. J. Radial. Oncol. Biol. Phys., 17: 691-694, 1989.
4. Hong. W. K.. Endicott. J., Uri. L. M.. Doos, W., Batsakis, J. G.. Bell, R., Fofonoff.
S.. Byers. R.. Atkinson. E. N.. Vaughan. C., Toth, B. B., Kramer, A.. Dimery, I. W.,
Skipper. P.. and Strong. S. 13-iÃ-Ã--Retinoicacid in the treatment of oral leukoplakia.
N. Engl. J. Med.. 315: 1501-1505. 1986.
5. Hong. K. W.. Lippman, S. M.. Uri. L. M.. Karp. D. D.. Lee. J. S.. Byers. R. M..
Schantz. S. P.. Kramer. A. M.. Lotan. R.. Peters. L. J.. Dimery. 1. W.. Brown. B. W.,
and Goepfert. H. Prevention of second primary tumors with isotretinoin in squamous
cell carcinoma of the head and neck. N. Engl. J. Med., 323: 795-801, 1990.
6. Lippman. S. M.. Batsakis. J. G.. Toth. B. B.. Weber. R. S.. Lee. J. J.. Martin. J. W..
Hays. G. L.. Goepfert. H.. and Hong. K. W. Comparison of low-dose isotretinoin w ith
ßcarotene to prevent oral carcinogenesis. N. Engl. J. Med., 32K: 15-20. 1993.
5557
Downloaded from cancerres.aacrjournals.org on July 12, 2017. © 1998 American Association for Cancer Research.
RETINOIDS.
INTERFERONS,
ANOIOOENESIS.
7. Amos. B.. and Lotan. R. Relinoid-sensiiive cells and cell lines. Methods Enzymol.,
190: 217-225. 1990.
8. Zou. C. P.. Clifford. J.. Xu, X. C.. Sacks. P.. Chambón, P.. Hong, W. K.. and Lotan,
R. Modulation by retinoic acid of squamous differentiation, cellular RA-binding
proteins, and nuclear RA receptors in human head and neck squamous carcinoma cell
lines. Cancer Res.. 54: 5479-5487, 1994.
9. Shalinsky. D. R.. Bischoff, E. D., Gregory, M. L„Goltardis, M. M., Hayes, J. S.,
Lamph, W. W., Heyman. R. A., Shirley. M. A.. Cooke, T. A., Davies, P. J.. and
Thorna/y. V. Retinoid-induced suppression of squamous cell differentiation in human
oral squamous cell carcinoma xenografts (line 1483) in athymic nude mice. Cancer
Res., 55: 3183-3191. 1995.
10. Lotan, R., Lotan. D., and Sacks. P. G. Inhibition of tumor cell growth by retinoids.
Methods Enzymol.. 190: 100-110. 1990.
11. Sacks, P. G., Harris. D., and Chou. T. C. Modulation of growth and proliferation in
squamous cell carcinoma by retinoic acid: a rationale for combination therapy with
chemotherapcutic agents. Int. J. Cancer. 61: 409-415. 1995.
12. Oikawa. T., Hirolani. K.. Nakamura. O.. Shudo. K.. Hiragun. A., and Iwahuchi, T. A
highly potent anti-angiogenic activity of retinoids. Cancer Lett., 48: 157-162. 1989.
13. Ingber. D.. and Folkman. J. Inhibition of angiogenesis through modulation of collagen
metabolism. Lab. Invest.. 59: 44-51, 1989.
14. Arensman, R. M.. and Stolar. C. H. Vitamin A effect on tumor angiogenesis.
J. Pediatr. Surg., 14: 809-813, 1979.
15. Majewski. S.. Szmurlo. A.. Marczak. M.. Jablonska. S., and Bollag. W. Inhibition of
tumor cell-induced angiogenesis by retinoids, 1.25-dihydroxyvitamin D3 and their
combinations. Cancer Lett.. 75: 35-39. 1993.
16. Lingen. M. W.. Polverini. P. J.. and Bouck. N. P. Inhibition of squamous cell
carcinoma angiogenesis by direct interaction of retinoic acid with endothelial cells.
Lab. Invest., 74: 476-483, 1996.
17. Lingen. M. W.. Polverini. P. J.. and Bouck. N. P. Retinoic acid induces cells cultured
from oral squamous cell carcinomas to become anti-angiogenic. Am. J. Pathol., 149:
247-258, 1996.
IX. Borden, E. C. Interferons: expanding therapeutic roles. N. Engl. J. Med., 326:
1491-1493, 1992.
19. Brouty-Boye. D., and Zeller, B. R. Inhibition of cell motility by interferon. Science
(Washington DC). 20«:516-518. 1980.
20. Sidky. Y., and Borden. E. Inhibition of angiogenesis by interferons: effects on tumor
and lymphiKyle-induced vascular responses. Cancer Res., 47: 5155-5161. 1987.
21. Friesel. R., Komoriya. A., and Maciag. T. Inhibition of endothelial cell proliferation
by gamma-interferon. J. Cell Biol.. 104: 689-696. 1987.
22. Maheshwari. R. K.. Srikantan, V., Bhartiya, D., Kleinman. H. K., and Grant. D. S.
Differential effects of interferon gamma and alpha on m vitro model of angiogenesis.
J. Cell Physio!.. 146: 164-169. 1990.
23. Ruszcak. Z., Detmar. M.. Imcke. E.. and Orfanos. C. E. Effects of rlFN alpha, beta
and gamma on the morphology, proliferation, and cell surface antigen expression of
human dermal microvascular endothelial cell in vitro. J. Invest. Dermatol.. 95:
693-699, 1990.
24. Saiki, I.. Sato, K., Yoo, Y. C.. Murata. J.. Yoneda, J.. Kiso, M., Hasegawa, A., and
Azuma, I. Inhibition of tumor-induced angiogenesis by the administration of recom
binant interferon-y followed by a synthetic lipid-A subunit analogue. Int. J. Cancer,
SI: 641-645. 1992.
25. Singh. R. K.. Gutman. M.. Bucana. C. D.. Sanchez. R.. Llansa. N.. and Fidler. I. J.
Interferons a and ßdown-regulate the expression of basic fibroblast growth factor in
human carcinomas. Proc. Nati. Acad. Sci. USA. 92: 4562-4566, 1995.
26. Ezekowitz, R. A., Mulliken. J. B., and Folkman, J. Interferon a-2a therapy for
life-threatening hemangiomas of infancy. N. Engl. J. Med.. 326: 1456-1463. 1992.
27. Jones, G. J., and Itri. L. Safety and tolerance of recombinant inlerferon a-2a in cancer
patients. Cancer (Phila.l. 57: 1709-1715, 1986.
28. Lippman. S. M.. Glisson, B. S., Kavanagh. J. J.. Lotan. R.. Hong. W. K.. ParedesEspinoza. M., l Im.'Im.ni. W. N.. Holdener. E. E.. and Krakoff. I. H. Retinoic acid and
inlerferon combination studies in human cancer. Eur. J. Cancer. 29A: 9-13. 1993.
29. Marth. C.. Daxenbichler, G., and Dapuni, O. Synergistic antiproliferative effect of
human recombinanl interféronsand retinoic acid in cultured breasl cancer cells.
J. Nail. Cancer Insl.. 77: 1197-1202. 1986.
30. Lolan. R.. Dawson. M. I., Zou, C. C.. Jong. L.. Lotan. D., and Zou, C. P. Enhanced
efficacy of combinations of retinoic acid and retinoid X receptor selective retinoids
and a-interferon in inhibition of cervical carcinoma cell proliferation. Cancer Res..
55: 232-236, 1995.
31. Kolla, V., Lindner, D. J.. Weihua. X., Borden. E. C., and Kalvakolanu. D. V.
Modulation of interferon-inducihle gene expression by retinoic acid: upregulalion of
stall protein in IFN-unresponsive cells. J. Biol. Chem.. 27/: 10508-10514. 1996.
32. Lindner, D. J., Borden, E. C„and Kalvakolanu. D. V. Synergistic antitumor effects
of a combination of interféronsand retinoic acid on human tumor cells in vitro and
in vivo. Clinical Cancer Res.. 3: 931-937. 1997.
AND ORAL CANCER
33. Toma. S.. Monteghirfo. S.. Tasso. P.. Nicolo. G.. Spadini. N., Palumbo, R., and
Molina. F. Antiproliferalive and Synergistic effect of interferon a-2a. and retinoids
and their association in established human cancer cell lines. Cancer Lett., 82:
209-216, 1994.
34. Frey. J.. Peck. R., and Bollag. W. Antiproliferative effects of retinoids. cylokines and
the combination in four human transformed epithelial cell lines. Cancer Lett.. 62:
167-172, 1992.
35. Moore, D. M., Kalvakolanu, D. V.. Lippman. S. M., Kavanagh, J. J., Hong, W. K.,
Borden, E. C., Paredes-Espinoza, M., and Krafoff, 1. H. Retinoic acid and inlerferon
in human cancer: mechanistic and clinical studies. Semin. Hematol..31: 31-37, 1994.
36. Boukamp. P.. Petrussevska, R. T., Breitkreulz, D., Hornung. J.. Markham, A., and
Fusenig, N. E. Normal keralinization in a spontaneously immortalized aneuploid
human keralinocyte cell line. J. Cell. Biol., 106: 761-771, 1988.
37. Rehberg, E., Kelder, B.. Hoal. E. G.. and Peslka, S. Specific molecular activities of
recombinant and hybrid leukocyte interferons. J. Biol. Chem.. 257: 11497-11502.
1982.
38. dayman, G. L.. EI-Naggar, A. K.. Zhang. W. W., Taylor. D. L.. Roth. J. A.. Goepfert,
H., and Liu. T-J. in vivo molecular therapy with p53 adenovirus for microscopic
residual head and neck squamous carcinoma. Cancer Res.. 55: 1-6, 1995.
39. Vermeulen. P. B.. Gasparini. G., Fox. S. B., Toi, M., Martin, L., McCullouch, P.,
Pezzella, F., Viale, G.. Weidner. N., Harris. A. L., and Dirix. L. Y. Quantification of
angiogenesis in solid human tumors: an international consensus on the methodology
and criteria of evalualion. Eur. J. Cancer. 32A: 2474-2484. 1996.
40. Oliveira, 1. C.. Sciavolino. P. J., Lee. T. H.. and Vilcek. J. Downregulation of
interleukin X gene expression in human fibroblasts: unique mechanism of transcriplional inhibilion by inlerferon. Proc. Nail. Acad. Sci. USA. 89: 9049-9053, 1992.
41. Oliveira, I. C.. Mukaida. M.. Matushima. K., and Vilcek. J. Transcriptional inhibilion
of Ihe IL-8 gene by interferon is mediated by Ihe NF-KB site. Mol. Cell. Biol.. 14:
5300-5308, 1994.
42. Shiro. M., and Matsushima, K. Enhanced phosphorylation of 65 and 74 kDa proteins
by lumor necrosis faclor a and IL-1 in human peripheral blood monocyles. Cytokine.
2: 13-20, 1990.
43. Larsen. C. G.. Kristensen. M.. Paludan, K., Deleuran. B., Thomsen. M. K., Zachariae.
K.. Malsushima. K., and Thestrup-Pederson. K. Vitamin D3 is a potenl regulator of
IL-1 induced IL-8 expression and production. Biochem. Biophys. Res. Commun..
176: 1020-1026. 1991.
44. Standiford. T. J.. Slreiler. R. M.. Chensue, S. W., Westwick. J., Kasahara, K., and
Kunkel. S. L. IL-4 inhibits Ihe expression of IL-8 from slimulaled human monocytes.
J. Immunol., 145: 1435-1439. 1990.
45. Anttila. H. S.. Reitamo. S.. Ceska. M.. and Hurme. M. Signal transduclion pathways
leading lo the production of IL-8 by human monocytes are differentially regulated by
dexamethasone. Clin. Exp. Immunol., 89: 509-512. 1992.
46. Brem, H., Gresser, I., Grosfeld. J., and Folkman. J. The combinalion of anliangiogenic agenls lo inhibit primary tumor growth and metaslasis. J. Pediatr. Surg.,
10: 1253-1257.
47. Parangi. S.. O'Reilly, M., Christofori, G.. Holmgrem, L., Grosfeld, J.. Folkman, J.,
48.
49.
50.
51.
52.
53.
and Hanahan. D. Anti-angiogenic therapy of transgenic mice impairs de nom lumor
growth. Proc. Nati. Acad. Sci. USA. 93: 2002-2007. 1996.
Bouck, N.. Stellmach, V.. and Hsu, S. How lumors become angiogenic. Adv. Cancer
Res.. 69: 135-174. 1996.
Hanahan. D.. and Folkman, J. Pallerns and emerging mechanisms of the angiogenic
switch during lumorigenesis. Cell. 86: 353-364. 1996.
Hori, A., Sadada, R., Masulani, E.. Nailo. K.. Sakura, Y.. Fujila. T.. and Kozai. Y.
Suppression of solid tumor growth by immunoneulralizing monoclonal anlibody
against human fibroblast growih faclor. Cancer Res.. 51: 6180-6184, 1991.
Kim, D. J.. Li, B., Winer, J.. Armanini, M.. Gillel. N., Phillips, H. S., and Ferrara, N.
Inhibilion of vascular endolhelial growth factor-induced angiogenesis suppresses
lumor cell growih in vii1».Nalure (Lond.l. 362: 841-844. 1993.
Millauer, B., Shawver, L. K.. Plate, K. H., Risau, W.. and Ullirich. A. Glioblastoma
growth inhibited in WTOby a dominant-negative Flk-l mm,mi Nalure (Lond.), 367:
576-578, 1994.
Holmgren, L., O'Reilly, M. S.. and Folkman. J. Dormancy of micromelaslases-
balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat.
Med.. /: 149-153, 1995.
54. O'Reilly, M. S., Holgren, L., Shing, Y., Chen. C.. Rosenthal. R. A., Moses, M., Lane,
W. S.. Cao,
inhibilor lhal
79: 315-328,
55. O'Reilly, M.
Y., Sage. E. H., and Folkman. J. Angiostalin: a novel angiogenesis
mediales Ihe suppression of métastasesby a Lewis lung carcinoma. Cell,
1995.
S.. Boehm, T.. Sing. Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E.,
Birkhead, J. R., Olsen. B. R.. and Folkman. J. Endostalin: an endogenous inhibilor of
angiogenesis and lumor growih. Cell, 88: 277-285. 1997.
5558
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Retinoic Acid and Interferon α Act Synergistically as
Antiangiogenic and Antitumor Agents against Human Head and
Neck Squamous Cell Carcinoma
Mark W. Lingen, Peter J. Polverini and Noël P. Bouck
Cancer Res 1998;58:5551-5558.
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