| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
Departments of Cancer Biology [K. I., J. W. S, S. J. K., B. Y. E., M. B-E., R. R., C. P. N. D.] and Urology [P. P., C. P. N. D.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Metastasis is a highly selective process involving multiple tumor-host interactions (4, 5, 6, 7) . A crucial step in metastasis is vascularization in and around the tumor (8, 9, 10) . This process of angiogenesis is regulated by the balance between stimulatory and inhibitory factors released by the tumor and the microenvironment (11) . Human bladder cancer produces a number of proangiogenic factors, including VEGF (12 , 13) , bFGF (14, 15, 16) , midkine (17) , thymidine phosphorylase (18) , and IL-8 (19) . MVD, a pathological surrogate for angiogenesis, correlates with stage and prognosis for patients with bladder cancer (20) .
IL-8 was originally identified as a leukocyte chemoattractant (21 , 22) , but subsequent studies have shown that IL-8 induces angiogenesis (23 , 24) and is expressed by melanoma (25, 26, 27) , lung (28) , prostate (29) , gastric (30) , and ovarian (31) cancers and by TCC of the bladder (19) . Although IL-8 is expressed by TCC, its exact role in the process of angiogenesis and the progression of TCC has not been elucidated.
Therefore, in the present study, we used an orthotopic model of human TCC to determine whether IL-8 regulates angiogenesis, tumorigenicity, and metastasis in TCC of the bladder.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Transfection and Selection of Tumor Cells Expressing IL-8.
The tumor cells were plated onto 100-mm dishes at a density of
1 x 106/dish. The monolayers
(60–70% confluent) were transfected with a full-length
pcDNA3/sense-IL-8, pcDNA3/AS-IL-8, or control pcDNA3 plasmids
[EcoRI-EcoRI, 1.5 kb; a gift from Dr. K.
Matsushima, University of Kanazawa, Kanazawa, Japan (21)
]
containing a drug-selectable marker for neomycin resistance and a
strong cytomegalovirus early promoter using a stable mammalian
transfection kit from Stratagene (La Jolla, CA). The cultures were
placed in a 37°C incubator for 12 h and then washed and fed with
CMEM. After 24 h, 500-1000 µg/ml G418 sulfate (Life
Technologies, Inc., Gaithersburg, MD) were added. The CMEM/G418 medium
was replaced every 3 days until individual, resistant colonies were
isolated and established in culture as individual lines. All cell lines
were maintained in CMEM/G418 and frozen after one to three in
vitro passages. The expression of IL-8 in individual clones was
identified by Northern blot analysis and ELISA. To avoid clonal
variations, positive clones were then pooled for the in
vitro and in vivo studies.
The 253J-P and 253J B-V cells were transfected with pcDNA3/sense IL-8 and pcDNA3/AS IL-8, respectively, or with control pcDNA3 plasmids. Individual G418-resistant (500–1000 µg/ml) colonies were established as separate adherent cultures. We selected pooled sense-IL-8-transfected 253J-P cells [253J-P(IL-8)], the highest IL-8-expressing 253J-P clone [253J-P(IL-8 High)], and the lowest IL-8-expressing 253J-P clone [253J-P(IL-8 Low)] and pooled AS-IL-8-transfected 253J B-V cells [253J B-V(AS IL-8)], the highest IL-8-expressing AS clone [253J B-V(AS IL-8 High)], and the lowest IL-8-expressing AS clone [253J B-V(AS IL-8 Low)], as indicated by the expression level of IL-8 mRNA and protein as determined by Northern blot analysis and ELISA, respectively.
Northern Blot Analysis.
Polyadenylated mRNA was extracted directly from the tumors or from
108 cultured cells using the Fasttrack mRNA
isolation kit (Invitrogen Co., San Diego, CA). The mRNA was
electrophoresed onto 1% denatured formaldehyde agarose gel,
electrotransferred to Genescreen nylon membranes (DuPont Co., Boston,
MA), and cross-linked with a UV Stratalinker 1800 (Stratagene, La
Jolla, CA) at 120,000 mJ/cm2. Filters were washed
twice at 65°C with 30 mM NaCl, 3 mM sodium
citrate, and 0.1% SDS (w/v). The membranes were then hybridized and
probed for IL-8, bFGF, VEGF, and MMP-9; the presence of GAPDH was used
to control for loading. The cDNA probes used were: a 0.5-kb
EcoRI cDNA fragment corresponding to human IL-8 (a gift of
Dr. K. Matsushima, University of Kanazawa, Kanazawa, Japan; Ref.
21
); a 1.4-kb cDNA fragment of bovine bFGF
(33)
; a 204-kb fragment of human VEGF cDNA inserted
in a pGEM-based construct (a gift of Dr. B. Berse, Harvard Medical
School, Boston, MA; Ref. 34
); a 1.0-kb cDNA
fragment corresponding to human MMP-9 (29)
; and a 1.28-kb
fragment from pR GAPDH cut with PstI (35)
. The
insert was excised with BamHI and EcoRI. Each
cDNA fragment was purified by agarose gel electrophoresis, recovered
using GeneClean (BIO 101, Inc., La Jolla, CA), and radiolabeled by a
random primer technique using a commercial kit (Boehringer Mannheim
Corp., Indianapolis, IN) and
[
-32P]deoxycytidine triphosphate (Amersham
Corp., Arlington Heights, IL; Ref. 36
). The steady-state
expressions of IL-8, bFGF, VEGF, and MMP-9 mRNA transcripts were
quantified by densitometry of autoradiographs with the use of the
ImageQuant software program (Molecular Dynamics, Sunnyvale, CA); each
sample measurement was calculated as the ratio of the average areas of
the specific mRNA transcripts to the 1.3-kb GAPDH mRNA transcript in
the linear range of the film.
Assay for IL-8, bFGF, and VEGF.
Viable cells (5 x103) were seeded in a 96-well
plate. Conditioned medium was removed after 24 h. The cells were
then washed with 200 µl of HBSS, and 200 µl of 10% bovine serum
supplemented by fresh MEM were added. Twenty-four h later, IL-8
and VEGF in cell-free culture supernatants and cell-associated bFGF in
freeze-thaw cell lysates were determined using the commercial Quantine
ELISA kit (R&D System, Minneapolis, MN). The protein concentration for
each factor was then determined by comparing the absorbance with that
of the standard. Results were expressed in terms of cell numbers
(37)
.
Growth Curve.
Viable cells (1 x103) were seeded in a 96-well
plate. Conditioned medium was removed after 24 h, the cells were
washed with 200 µl of HBSS, and 200 µl of fresh CMEM or CMEM/G418
conditioned medium were added. Every 24 h, the number of viable
cells in each cell line was determined by absorbance comparison. The
doubling times of each cell line were determined by plotting the
absorbance on a semilogarithmic axis versus time (Cricket
Software, Malvern, PA; Fig. 3
). The doubling times of the 253J-P
sense-IL-8 transfectants (IL-8, 35.0 h; IL-8 Low, 37.2 h; and
IL-8 High, 37.6 h) were similar to those for both 253J-P (35.0 h)
and 253J-P(Neo) (35.4 h), and the doubling times of the 253J B-V
AS-IL-8 transfectants (AS IL-8, 26.6 h; AS IL-8 Low, 27.3 h;
and AS IL-8 High, 27.1 h) were similar to 253J B-V (26.0 h) and
253J B-V(Neo) (26.9 h).
|
We next investigated whether the increase in MMP-9 activity is mediated by IL-8. Parental 253J-P cells were incubated in the presence of different doses of (0–20 µg/ml of human rIL-8), and the activity of MMP-9 was determined. Next, we investigated whether the increased activity of MMP-9 caused by rIL-8 was inhibited by neutralization with an anti-IL-8 antibody (100 µg/ml), with nonspecific IgG (100 µg/ml) serving as control.
PCR Analysis.
RT-PCR analysis was performed as described previously
(39)
. Briefly, total cellular RNA (1 mg) extracted from
various cell lines was transcribed into cDNA using downstreaming
primers IL-8 receptor type A and IL-8 receptor type B, respectively
(Reverse Transcription System; Promega Corp., Madison, WI). The reverse
transcription reaction was performed at 42°C for 50 min. PCR was
performed with 40 cycles of denaturation (94°C for 1.5 min),
annealing (58°C for 45 s), extension (72°C for 2.5 min), and 7
min of extension after completion of all cycles. Amplified fragments
were analyzed on the 2% gel, and bands of expected sizes were
confirmed by sequencing. The primer sequences used were as follows:
IL-8 receptor type A, sense 5'-AGT TCT TGG CAC GTC ATC G-3' and AS
5'-CTT GGA GGT ACC TCA ACA GC-3'; and IL-8 receptor type B, sense
5'-ACA TTC CTG TGC AAG GTG G-3' and AS 5'-CAG GGT GAA TCC GTA GCA
GA-3'.
Invasion through Matrigel.
Polyinylprolidone-free polycarbonate filters (8-µm pore size;
Nucleopore; Becton Dickinson Labware, Franklin Lakes, NJ) were coated
with a mixture of basement membrane components (Matrigel; 25
µg/filter) and placed in modified Boyden chambers. The cells
(2 x 105) were released from
their culture dishes by short exposure to EDTA (1 mM),
centrifuged, resuspended in 0.1% BSA/DMEM, and placed in the upper
compartment of the Boyden chamber. Fibroblast-conditioned medium was
placed in the lower compartment as a source of chemoattractants. After
incubation for 6 h at 37°C, the cells on the lower surface of
the filter were stained with Diff-Quick (American Scientific Products,
McGaw Park, IL) and quantified with a cooled charge-coupled device
Optotronics Tec 470 camera (Optotronics Engineering, Goletha, CA)
linked to a computer and digital printer (Sony Corp., Tokyo, Japan).
The results were expressed as the average number of cells in the five
highest spots identified within a single x200 field on the lower
surface of the filter (40)
.
Animals.
Male athymic BALB/c nude mice were obtained from the Animal Production
Area of the National Cancer Institute, Frederick Cancer Research
Facility (Frederick, MD). The mice were maintained in a laminar airflow
cabinet under specific pathogen-free conditions and used at 8–12 weeks
of age. All facilities were approved by the American Association for
Accreditation of Laboratory Animal Care in accordance with the current
regulations and standards of the United States Department of
Agriculture, the Department of Health and Human Services, and the NIH.
Orthotopic Implantation of Tumor Cells.
Cultured 253J-P, 253J B-V, Neo-transfected, and sense- and
AS-IL-8-transfected cells (60–70% confluent) were prepared for
injection as described previously (32)
. Mice were
anesthetized with methoxyflurane. For orthotopic implantation, a lower
midline incision was made, and viable tumor cells (2 x 106 in 0.05 ml) of HBSS were implanted into the
bladder wall. The formation of a bulla indicated a satisfactory
injection. The bladder was returned to the abdominal cavity, and the
abdominal wall was closed with a single layer of metal clips. The mice
were killed and necropsied 6 weeks after implantation of tumor cells.
The primary tumors were removed and weighed, and the presence of
metastases (in lymph nodes and lung) was determined grossly and
microscopically. The bladders were then either quickly frozen in liquid
nitrogen for mRNA extraction, fixed in 10% buffered formalin, placed
in OCT compound (Miles Laboratories, Elkhart, IN), or mechanically
dissociated and put into tissue culture.
In Situ mRNA Hybridization Analysis.
Specific AS oligonucleotide DNA probes were designed complementary to
the mRNA transcripts based on published reports of the cDNA sequence:
IL-8, CTC CAC AAC CCT CTG CAC CC, 66% GC content (21)
;
bFGF, CGG GAA GGC GCC GCT GCC GCC, 85.7% GC content (33)
;
VEGF/VPF, TGG TGA TGT TGG ACT CTT CAG TGG GCU, 57.7% GC content
(34)
; and MMP-9, CCG GTC CAC CTC GCT GGC GCT CCG GU,
80.0% GC content (29)
. The specificity of the
oligonucleotide sequence was initially determined by a Gene Bank
European Molecular Biology Library database search with the use of the
Genetics Computer Group sequence analysis program (Genetics Computer
Group, Madison, WI), based on the FastA algorithm; these sequences
showed 100% homology with the target gene and minimal homology with
nonspecific mammalian gene sequences. The specificity of each of the
sequences was also confirmed by Northern blot analysis
(41)
. A poly d(T)20 oligonucleotide
was used to verify the integrity and lack of degradation of mRNA in
each sample. All DNA probes were synthesized with six biotin molecules
(hyperbiotin-ylated) at the 3' end via direct coupling, with the
use of standard phosphor-amidite chemical methods (Research
Genetics, Huntsville, AL). The lyophilized probes were reconstituted in
a stock solution at 1 µg/µl in 10 mM Tris (pH 7.6) and
1 mM EDTA. Immediately before use, the stock solution was
diluted with probe dilution (Research Genetics).
ISH mRNA hybridization was performed as described previously with minor modifications (42 , 43) . ISH was performed using the Microprobe Manual Staining System (Fisher Scientific, Pittsburgh, PA; Ref. 44 ). Tissue sections (4 µm) of formalin-fixed, paraffin-embedded specimens were mounted on silane-treated ProbeOn slides (Fisher Scientific; Refs. 42 and 43 ). The slides were placed in the Microprobe slide holder, dewaxed, and rehydrated with Autodewaxer and Autoalcohol (Research Genetics), followed by enzymatic digestion with pepsin. Hybridization of the probe was performed for 45 min at 45°C, and the samples were then washed three times with 2x SSC for 2 min at 45°C. The samples were incubated with alkaline phosphatase-labeled avidin for 30 min at 45°C, rinsed in 50 mM Tris buffer (pH 7.6), rinsed with alkaline phosphatase enhancer for 1 min, and incubated with a chromogen substrate for 15 min at 45°C. If necessary, samples were incubated a second time with fresh chromogen substrate to enhance a weak reaction. A red stain indicated a positive reaction. To control for endogenous alkaline phosphatase, the sample was treated in the absence of the biotinylated probe, using chromogen alone.
Quantification of Color Reaction.
Stained sections were examined in a Zeiss photomicroscope (Carl Zeiss,
Thornwood, NY) equipped with a three-chip, charge-coupled device color
camera (model DXC-969 MD; Sony Corp.). The images were analyzed using
the Optimas image analysis software (version 4.10; Bothell, WA). The
slides were prescreened by one of the investigators to determine the
range in staining intensity of the slides to be analyzed. Images
covering the range of staining intensities were captured
electronically, a color bar (montage) was created, and a threshold
value was set in the red, green, and blue mode of the color camera. All
subsequent images were quantified based on this threshold. The
integrated absorbance of the selected fields was determined based on
its equivalence to the mean log inverse gray value multiplied by the
area of the field. The samples were not counterstained; therefore, the
absorbance was attributable solely to the product of the ISH reaction.
Three different fields in each sample were quantified to derive an
average value. The intensity of staining was determined by comparison
with the integrated absorbance of poly d(T)20.
The results were presented as the number of each cells for line
compared with the control, which was set to 100 (37)
.
Immunohistochemical Analysis.
For immunohistochemical analysis, frozen tissue sections (8-µm thick)
were fixed with cold acetone. Tissue sections (5-µm thick) of
formalin-fixed, paraffin-embedded specimens were deparaffinized in
xylene, rehydrated in graded alcohol, and transferred to PBS. The
slides were rinsed twice with PBS, and antigen retrieval was performed
with pepsin for 12 min; endogenous peroxidase was blocked by the use of
3% hydrogen peroxide in PBS for 12 min. The samples were washed three
times with PBS and incubated for 20 min at room temperature with a
protein-blocking solution of PBS (pH 7.5) containing 5% normal horse
serum and 1% normal goat serum. Excess blocking solution was drained,
and the samples were incubated for 18 h at 4°C with a 1:100
dilution of rat monoclonal anti-CD31 antibody (PharMingen, San Diego,
CA; Ref. 45
), a 1:50 dilution of a rabbit polyclonal
anti-IL-8 antibody (Biosource International, Camarillo, CA), a 1:500
dilution of rabbit polyclonal anti-bFGF antibody (Sigma Chemical Co.,
St. Louis, MO), a 1:500 dilution of rabbit polyclonal anti-VEGF/VPF
antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or a 1:100
dilution of mouse monoclonal anti-MMP-9 antibody (Oncogene Research
Products, Cambridge, MA). The samples were then rinsed four times with
PBS and incubated for 60 min at room temperature with the appropriate
dilution of the secondary antibody:peroxidase-conjugated antirat IgG
(H+L; Jackson ImmunoResearch Laboratory, Inc., West Grove, PA),
antirabbit IgG, F(ab)2 fragment (Jackson
ImmunoResearch Laboratory, Inc.), or antimouse IgG1 (PharMingen). The
slides were rinsed with PBS and incubated for 5 min with
diaminobenzidine (Research Genetics). The sections were then washed
three times with PBS, counterstained with Gill’s hematoxylin (Biogenex
Laboratories, San Ramon, CA), and washed three times with PBS. The
slides were mounted with Universal Mount mounting medium (Research
Genetics).
Quantification of MVD.
MVD was determined by light microscopy after immunostaining of sections
with anti-CD31 antibodies according to the procedure of Weidner
et al. (46)
. Clusters of stained endothelial
cells distinct from adjacent microvessels, tumor cells, or other
stromal cells were counted as one microvessel. The tissue was recorded
using a cooled charge-coupled device Optotronics Tec 470 camera
(Optotronics Engineering) linked to a computer and digital printer
(Sony Corp.). The density of microvessels was expressed as the average
number of the five highest areas identified within a single x200
field.
Quantification of Intensity of Immunostaining.
The intensity of immunostaining of IL-8, bFGF, VEGF, and MMP-9 was
quantified in three different areas of each sample by an image analyzer
using the Optimas software program (Bioscan, Edmonds, WA). Three
different areas in each sample were quantified to yield an average
measurement of intensity of immunostaining. The results were presented
as the number of cells for each cell line compared with the control,
which was set to 100 (37)
.
MMP-9 mRNA Half-Life Studies.
To determine the effect of IL-8 on MMP-9 mRNA stability, 253J-P, 253J
B-V, 253J-P(Neo), 253J B-V(Neo), 253J-P(IL-8), and 253J B-V(AS IL-8)
cells were incubated for 24 h. Further transcription in cells was
then blocked by the addition of ActD (Calbiochem-Novabiotechnology,
Inc., Lake Placid, NY; final concentration, 5 µg/ml). Total RNA was
extracted from the cells 0, 0.5, 1, 2, 4, and 6 h after the
addition of ActD, and Northern blot analysis was performed for MMP-9
mRNA expression. MMP-9 mRNA expression of each time point was compared
with the control value (total RNA extracted from cells prior to ActD
treatment was arbitrarily defined as 100%). The half-life of MMP-9
mRNA was determined by plotting relative MMP-9 mRNA expression levels
on a semilogarithmic axis versus time (Cricket Software,
Malvern, PA).
CAT Assay.
Using the FuGENE 6 protocol (Boehringer Mannheim Corp.), we transfected
the basic CAT expression vector with no promoter/enhancer sequences
(pCAT-basic) or a control plasmid with SV40 promoter and enhancer
(pCAT-control; Promega) into 253J-P cells, sense-transfected cells,
253J-BV cells, AS-transfected cells, and each Neo transfectant. One
copy of the full-sequence, human 570-bp MMP-9 promoter (a gift of Dr.
Seiki Motoharu, University of Tokyo, Tokyo, Japan) was ligated
upstream of the basic CAT expression vector. We transfected 5 x 103 cells/well of six-well tissue culture
dishes with 2.5 µg of the reporter CAT constructs and 2.5 µg of a
ß-actin expression plasmid. After 48 h, extracts were prepared
from all plates, normalized for ß-actin activity, and assayed for CAT
activity (47)
according to the methods of Hudson et
al. (48)
, described previously. The CAT assay was
quantified by densitometry of autoradiographs with the use of the Image
Quant software program (Molecular Dynamics, Sunnyvale, CA) and was
evaluated as the ratio of acetylated species to all species.
Statistical Analysis.
The statistical differences in vessel counts and staining intensity for
IL-8, bFGF, VEGF, and MMP-9 of bladder tumors were analyzed by the
Mann-Whitney U test. The incidences of tumor and metastasis
were statistically analyzed by Fisher’s exact test. A value of
P < 0.05 was considered significant.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Northern blot analyses for the highly metastatic human TCC
cell line 253J B-V; its AS-IL-8 transfectants 253J B-V(AS IL-8), 253J
B-V(AS IL-8 Low), and 253J B-V(AS IL-8 High); and the Neo-transfected
253J-BV(Neo) are shown in Fig. 1B
. The level of
expression is shown as the ratio of mRNA expression of the
transfectants to that of both the corresponding parental and Neo
transfectant cell lines. IL-8 expression was increased 3.3-, 4.6-, and
2.0-fold by the 253J-P(IL-8), 253J-P(IL-8 High), and the 253J-P(IL-8
Low) cell lines, respectively, whereas there was no change in the mRNA
expression of bFGF or VEGF. The mRNA expression of IL-8 by 253J B-V(AS
IL-8) and 253J B-V(AS IL-8 Low) was decreased 2.0- and 3.3-fold,
respectively, whereas there was no change in either bFGF or VEGF mRNA
expression. IL-8, bFGF, and VEGF protein production by 253J-P, 253J
B-V, and the transfected cell lines was evaluated by ELISA (Fig. 2)
. Changes in protein expression by the transfectants paralleled the
changes seen in mRNA expression. Levels of IL-8 expression were 3.5-
and 4.3-fold higher in the 253J-P(IL-8) and 253J-P(IL-8 High) cells,
respectively, and decreased 2.1- and 2.4-fold by the 253J B-V(AS IL-8)
and 253J B-V(AS IL-8 Low) cell lines. bFGF and VEGF protein expression
was unchanged.
|
|
shows that MMP-9 mRNA expression was increased 2.2- and 2.6-fold
by 253J-P(IL-8) and 253J-P(IL-8 High) cells, respectively, and reduced
2.5- and 3.3-fold by 253J B-V(AS IL-8) and 253J B-V(AS IL-8 Low),
respectively, after transfection with IL-8 sense or AS transcripts.
These results show that IL-8 regulates MMP-9 expression by the 253J-P
and 253J B-V human TCC cells.
Collagenase Activity.
To demonstrate that MMP-9 expressed by the transfected cells is
biologically active, collagenase activity was determined by zymogram
after normalizing the volume of supernatant for cell number (Fig. 3)
. By densitometry, the collagenase activity of 253J-P(IL-8) and
253J-P(IL-8 High) was increased 5.5- and 7.7-fold, respectively,
compared with 253J-P and 252J-P(Neo) (Fig. 4A)
, whereas that of 253J B-V(AS IL-8) and 253J B-V(AS IL-8
Low) was decreased 3.0- and 3.6-fold, respectively, compared with
either 253J B-V or 252J B-V(Neo) (Fig. 4B)
.
|
. To that end, parental 253J-P cells were incubated in the
presence of different doses of human rIL-8, and the activity of MMP-9
was determined by zymography after normalizing the volume of
supernatant for cell number. The results shown in Fig. 4A
indicate that IL-8 caused an increase in the activity of MMP-9.
Moreover, the increased activity of MMP-9 by rIL-8 was inhibited by
neutralization with anti-IL-8 antibody (Fig. 4B)
.
RT-PCR Analysis.
RT-PCR analysis revealed that 253J-P, 253J-P(Neo), and
sense-IL-8-transfected 253J-P and also 253J B-V, 253J B-V(Neo), and
AS-IL-8-transfected 253J B-V express mRNA for both types of IL-8
receptors (Fig. 5)
.
|
). Invasion by 253J B-V(AS IL-8) and 253J B-V(AS IL-8 Low)
was reduced 63 and 78%, respectively, compared with invasion by 253J
B-V or 253J B-V(Neo) (P < 0.005; Fig. 6B
).
|
CAT Activity.
The full sequence MMP-9 promoter was linked upstream of the CAT
reporter gene and transfected into sense-IL-8 transfected,
AS-IL-8-transfected, each Neo-transfected, and each parental cell to
examine the effect of IL-8 expression on MMP-9 transcription.
Forty-eight h after transfection, cell extracts were prepared, and
equivalent amounts of extracts exhibiting the same ß-actin activity
were tested for CAT activity. CAT activity driven by the MMP-9 promoter
in 253J-P(IL-8) and 253J-P(IL-8 High) was increased 1.4- and 1.8-fold,
respectively (Fig. 7A)
, compared with either 253J-P or 253J-P(Neo), and decreased
1.4- and 1.7-fold by 253J B-V(AS IL-8) and 253J B-V(AS IL-8 Low),
respectively (Fig. 7B)
, compared with either 253J B-V and
253J B-V(Neo). CAT activity driven by the SV-40 promoter was the same
in both cell populations and served as an additional internal control
for transfection efficiency.
|
. The 253J-P(IL-8) and 253J-P(IL-8 High) cells formed tumors that were
palpable at 6 weeks and demonstrated a significant enhancement of
tumorigenicity compared with either 253J-P or 253J-P(Neo). No tumors
were detected either grossly or microscopically in mice implanted with
253J-P or 253J-P(Neo) at 6 or 12 weeks. Three of 10 animals implanted
with 253J-P(IL-8) developed tumors with a median weight of 124 mg
(range, 25–303 mg; P < 0.05 compared with
both controls); and three of nine animals implanted with 253J-P(IL-8
High) developed tumors with a median weight of 161 mg (range, 26–364
mg; P < 0.05 compared with either control).
None of the mice implanted with 253J-P(IL-8 Low) developed tumors by 6
or 12 weeks. By 12 weeks, lymph node metastasis were observed in the
mice implanted with 253J-P(IL-8) and 253J-P(IL-8 High) cells.
|
. By 6 weeks, tumors were palpable in all
mice implanted with 253J B-V (median weight, 188 mg; range, 112–284
mg) and 253J B-V(Neo) (median weight, 237 mg; range, 88–516 mg).
Tumors also were detected in 9 of 10 mice implanted with 253J B-V(IL-8
High) (median weight, 105 mg; range, 26–333 mg). In contrast,
tumorigenicity was significantly reduced after implantation of the 253J
B-V(AS IL-8) and 253J B-V(AS IL-8 Low) cells. Only five of eight mice
implanted with 253J B-V(AS IL-8) developed tumors at 6 weeks, and these
tumors were significantly smaller than the 253J B-V or 253J B-V(Neo),
[median weight, 40 mg; range, 25–321 mg; P < 0.01 compared with 253J B-V and 253J B-V(Neo)]. Four of eight
mice implanted with 253J B-V(AS IL-8 Low) developed tumors by 6 weeks,
and these tumors were also significantly smaller than either 253J B-V
or 253J B-V [median weight, 40 mg; range, 27–72 mg;
P < 0.01 compared with 253J B-V or 253J
B-V(Neo)]. Furthermore, no spontaneous lymph node metastases were
detected in mice implanted with either 253J B-V(AS IL-8) or 252J B-V(AS
IL-8 Low) at 6 weeks [P < 0.05, compared
with either 253J B-V (four of eight) or 253J B-V(Neo) (eight of
nine)]. Therefore, IL-8 expression regulates both the tumorigenicity
and metastasis of human TCC.
Expression of IL-8, bFGF, VEGF, and MMP-9.
The in vivo expressions of IL-8, bFGF, VEGF, and MMP-9 mRNA
and protein were evaluated by ISH (Table 2
; Fig. 8
) and immunohistochemical staining (Table 2
; Fig. 9
), respectively, and correlated directly with the in vitro
expression of these factors. Both the 253J-P(IL-8) and 253J-P(IL-8
High) tumors expressed equal amounts of IL-8 and MMP-9 mRNA and
protein. Both IL-8 and MMP-9 expression was down-regulated in the AS
IL-8-transfected tumors. IL-8 mRNA expression was down-regulated 30 and
55%, and MMP-9 mRNA was down-regulated 29 and 32% in the 253J B-V(AS
IL-8) and 253J B-V(AS IL-8 Low) tumors, respectively. IL-8 protein
expression was likewise down-regulated 28 and 40%, and MMP-9 protein
was down-regulated 30 and 37% by 253J B-V(AS IL-8) and 253J B-V(AS
IL-8 Low), respectively. No difference was observed in the in
vivo expression of bFGF or VEGF mRNA or protein after transfection
with IL-8 sense or AS transcripts.
|
|
|
; Fig. 9
). The number of CD31+ microvessels counted per
x200 field in the 253J-P(IL-8) and 253J-P(IL-8 High) tumors was
65 ± 21 and 74 ± 25, respectively.
Because the 253J-P and 253J-P(Neo) cells did not develop tumors, we
could not evaluate the effect of the overexpression of IL-8 on
tumor-induced neovascularization. However, AS-IL-8 transfection
significantly inhibited tumor-induced neovascularization of the 253J
B-V cells. The MVD was significantly reduced from 116 ± 41 and 134 ± 50 in the 253J B-V and 253J B-V(Neo)
tumors, respectively, to 65 ± 12 and 47 ± 13 in the 253J B-V(AS IL-8) and 253J B-V(AS IL-8 High) bladder
tumors (P < 0.005; Table 2
; Fig. 9
).
Tumor-induced neovascularization correlates directly with the IL-8
expression of the tumors and tumor growth and metastasis. |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present study confirms and expands upon these studies. Our goal was to evaluate whether the level of expression of IL-8 directly regulates tumor-induced neovascularization and subsequent tumor growth and metastasis of human TCCs growing within the bladder of athymic nude mice. We enforced IL-8 expression by transfecting the poorly tumorigenic and nonmetastatic human TCC cell line 253J-P (which expresses relatively low levels of IL-8) with the sense-IL-8 construct and were able to establish several cell lines that overexpressed IL-8. Two of these cell lines, 253J-P(IL-8) and 253J-P(IL-8 High), demonstrated enhanced tumorigenicity and spontaneous lymph node metastasis compared with the 253J-P or 253J-P(Neo) controls. Conversely, after AS-IL-8 transfection, we were able to reduce IL-8 expression by the highly tumorigenic and metastatic 253J B-V cell line (which expresses relatively high levels of IL-8) and establish cell lines that were significantly less tumorigenic (lower incidence and smaller tumors) and metastatic than 253J B-V or 253J B-V(Neo) controls. Because neither bFGF nor VEGF expression was altered by IL-8 transfection, we conclude that these effects are independent of the activity of these angiogenesis factors. Furthermore, because IL-8 transfection did not affect in vitro proliferation of 253J-P or 253J B-V, the effects on growth and metastasis are independent of proliferation, although the cells do have both type A (CXCR1; Refs. 50 and 51 ) and type B (CXCR2; Refs. 50 and 51 ) IL-8 receptors. Therefore, our results provide direct evidence for the involvement of IL-8 in the induction of in vivo angiogenesis and in the subsequent growth and metastasis of TCC. These results are similar to previous reports in which transfection with VEGF or bFGF increased MVD and enhanced tumor growth and metastasis of melanoma and breast cancer (52, 53, 54) .
The metastatic potential of bladder cancer depends upon the expression of several metastasis-related genes, such as IL-8, that regulate endothelial cell proliferation and capillary morphogenesis (55) , and other genes, such as MMP-9, that regulate the degradation of the extracellular matrix (56, 57, 58, 59, 60, 61) . The local production of MMP-9 or other proteases such as plasminogen activator by bladder cancer cells or stroma facilitates the local degradation of the extracellular matrix and results in tumor invasion and subsequent metastasis (56, 57, 58, 59, 60, 61) . The proteolytic effect of MMPs facilitates the migration of endothelial cells through the altered extracellular matrix toward the source of the angiogenic stimulus; in this manner, MMPs are an integral component of the angiogenesis pathway. The highly metastatic 253J B-V cell line expresses high levels of the metalloproteinase MMP-9 compared with the nonmetastatic 253J-P cell line. Recently, Luca et al. (27) reported that IL-8 induces MMP-2 activity by malignant melanoma cells. The up-regulation of collagenase activity by IL-8 was considered to be an important mechanism to explain the associated increase in metastatic ability. Similarly, we found that the activity of MMP-9 by the TCC cells directly correlated with their expression of IL-8. Moreover, when we altered the expression of IL-8 by sense or AS transfection, we observed a corresponding change in MMP-9 expression and activity. The MMP-9 induced by transfection is biologically active, because it induced collagenase activity and increased cellular invasion through Matrigel; when it was reduced after AS transfection, both collagenase activity and invasion through Matrigel were decreased. When the 253J B-V(AS IL-8) and 253J B-V(AS IL-8 Low) cells were implanted in vivo, the expression of MMP-9 was reduced within the tumors. These tumors were smaller than their controls, which may reflect relative growth inhibition secondary to the inability to induce as robust a microcirculation, and were nonmetastatic, attributable perhaps to a reduction in MMP-9 activity.
It is likely that IL-8 regulates MMP-9 expression at the transcriptional level. To investigate this mechanism, we evaluated MMP-9 mRNA stability and the level of gene transcription of MMP-9 in IL-8 transfectants and control cells. Although the expression of MMP-9 mRNA varied among the IL-8 transfectants and controls, the stability of MMP-9 mRNA was not changed by transfection with sense-IL-8 or AS-IL-8. However, CAT activity driven by the MMP-9 promoter was up-regulated in IL-8 sense transfectants and down-regulated after AS transfection. It is well established that bFGF regulates MMP activity in TCC (62) . Because bFGF levels are not affected by IL-8 transfection, in our cells the regulation of MMP-9 transcription is independent of bFGF and likely attributable to the level of IL-8. These results are in keeping with the report of Luca et al. (27) , who found that IL-8 regulated MMP-2 gene transcription.
In summary, our present study demonstrates that IL-8 regulates angiogenesis, tumorigenesis, and metastasis by human TCC, which may be mediated in part by regulating the expression and activity of MMP-9.
|
FOOTNOTES |
|---|
1 Supported by NIH Grants CA67914 and CA56973 and
Core Grant CA16672. 
2 To whom requests for reprints should be
addressed, at Department of Cancer Biology, Box 173, The University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,
TX 77030. Phone: (713) 792-8165; Fax: (713) 792-8747; E-mail: cdinney{at}mdanderson.org
3 The abbreviations used are: TCC, transitional
cell carcinoma; VEGF, vascular endothelial cell growth factor; bFGF,
basic fibroblast growth factor; IL, interleukin; rIL, recombinant IL;
MVD, microvessel density; AS, antisense; MMP-9, matrix
metalloproteinase type 9; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; ActD, actinomycin D; CAT, chloramphenicol
acetyltransferase; RT-PCR, reverse transcription-PCR; CMEM, complete
Eagle’s minimum essential medium; ISH, in situ
hybridization.
Accepted 2/18/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. J.J. Waugh and C. Wilson The Interleukin-8 Pathway in Cancer Clin. Cancer Res., November 1, 2008; 14(21): 6735 - 6741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Lu, J. Wang, Y. Xu, A. E. Koch, Z. Cai, X. Chen, D. L. Galson, R. S. Taichman, and J. Zhang CXCL16 Functions as a Novel Chemotactic Factor for Prostate Cancer Cells In vitro Mol. Cancer Res., April 1, 2008; 6(4): 546 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F. MacManus, J. Pettigrew, A. Seaton, C. Wilson, P. J. Maxwell, S. Berlingeri, C. Purcell, M. McGurk, P. G. Johnston, and D. J.J. Waugh Interleukin-8 Signaling Promotes Translational Regulation of Cyclin D in Androgen-Independent Prostate Cancer Cells Mol. Cancer Res., July 1, 2007; 5(7): 737 - 748. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Huang, A. Lavoie-Lamoureux, K. Moran, and J.-P. Lavoie IL-4 stimulates the expression of CXCL-8, E-selectin, VEGF, and inducible nitric oxide synthase mRNA by equine pulmonary artery endothelial cells Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1147 - L1154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zhao, M.-J. Kwon, S. Huang, J. Y. Lee, K. Fukase, N. Inohara, and D. H. Hwang Differential Modulation of Nods Signaling Pathways by Fatty Acids in Human Colonic Epithelial HCT116 Cells J. Biol. Chem., April 20, 2007; 282(16): 11618 - 11628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Mitra, R. H. Datar, and R. J. Cote Molecular Pathways in Invasive Bladder Cancer: New Insights Into Mechanisms, Progression, and Target Identification J. Clin. Oncol., December 10, 2006; 24(35): 5552 - 5564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Myers-Irvin, T.-S. Van Le, and R. H. Getzenberg Mechanistic Analysis of the Role of BLCA-4 in Bladder Cancer Pathobiology Cancer Res., August 15, 2005; 65(16): 7145 - 7150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Doubrovina, M. M. Doubrovin, E. Vider, R. B. Sisson, R. J. O'Reilly, B. Dupont, and Y. M. Vyas Evasion from NK Cell Immunity by MHC Class I Chain-Related Molecules Expressing Colon Adenocarcinoma J. Immunol., December 15, 2003; 171(12): 6891 - 6899. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. De Larco, B. R. K. Wuertz, D. Yee, B. L. Rickert, and L. T. Furcht Atypical methylation of the interleukin-8 gene correlates strongly with the metastatic potential of breast carcinoma cells PNAS, November 25, 2003; 100(24): 13988 - 13993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Mian, C. P. N. Dinney, C. E. Bermejo, P. Sweeney, C. Tellez, X. D. Yang, J. M. Gudas, D. J. McConkey, and M. Bar-Eli Fully Human Anti-Interleukin 8 Antibody Inhibits Tumor Growth in Orthotopic Bladder Cancer Xenografts via Down-Regulation of Matrix Metalloproteases and Nuclear Factor-{kappa}B Clin. Cancer Res., August 1, 2003; 9(8): 3167 - 3175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Karashima, P. Sweeney, A. Kamat, S. Huang, S. J. Kim, M. Bar-Eli, D. J. McConkey, and C. P. N. Dinney Nuclear Factor-{kappa}B Mediates Angiogenesis and Metastasis of Human Bladder Cancer through the Regulation of Interleukin-8 Clin. Cancer Res., July 1, 2003; 9(7): 2786 - 2797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Lu, Y. Nakamoto, Y. Nemoto-Sasaki, C. Fujii, H. Wang, M. Hashii, Y. Ohmoto, S. Kaneko, K. Kobayashi, and N. Mukaida Potential Interaction between CCR1 and Its Ligand, CCL3, Induced by Endogenously Produced Interleukin-1 in Human Hepatomas Am. J. Pathol., April 1, 2003; 162(4): 1249 - 1258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Levy, C. Neuveut, C.-A. Renard, P. Charneau, S. Branchereau, F. Gauthier, J. T. Van Nhieu, D. Cherqui, A.-F. Petit-Bertron, D. Mathieu, et al. Transcriptional Activation of Interleukin-8 by beta -Catenin-Tcf4 J. Biol. Chem., October 25, 2002; 277(44): 42386 - 42393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, E. Gjernes, and H. Prydz Factor VIIa Induces Tissue Factor-dependent Up-regulation of Interleukin-8 in a Human Keratinocyte Line J. Biol. Chem., June 21, 2002; 277(26): 23620 - 23626. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Inoue, M. Kamada, J. W. Slaton, S. Fukata, C. Yoshikawa, P. Tamboli, C. P. N. Dinney, and T. Shuin The Prognostic Value of Angiogenesis and Metastasis-related Genes for Progression of Transitional Cell Carcinoma of the Renal Pelvis and Ureter Clin. Cancer Res., June 1, 2002; 8(6): 1863 - 1870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Feoktistov, A. E. Goldstein, S. Ryzhov, D. Zeng, L. Belardinelli, T. Voyno-Yasenetskaya, and I. Biaggioni Differential Expression of Adenosine Receptors in Human Endothelial Cells: Role of A2B Receptors in Angiogenic Factor Regulation Circ. Res., March 22, 2002; 90(5): 531 - 538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Van Coillie, I. Van Aelst, A. Wuyts, R. Vercauteren, R. Devos, C. De Wolf-Peeters, J. Van Damme, and G. Opdenakker Tumor Angiogenesis Induced by Granulocyte Chemotactic Protein-2 as a Countercurrent Principle Am. J. Pathol., October 1, 2001; 159(4): 1405 - 1414. [Abstract] [Full Text]
|
|
|
|
|
|
J. E. De Larco, B. R. K. Wuertz, J. C. Manivel, and L. T. Furcht Progression and Enhancement of Metastatic Potential after Exposure of Tumor Cells to Chemotherapeutic Agents Cancer Res., April 1, 2001; 61(7): 2857 - 2861. [Abstract] [Full Text]
|
|
|
|
|
|
K. Inoue, J. W. Slaton, T. Karashima, C. Yoshikawa, T. Shuin, P. Sweeney, R. Millikan, and C. P. N. Dinney The Prognostic Value of Angiogenesis Factor Expression for Predicting Recurrence and Metastasis of Bladder Cancer after Neoadjuvant Chemotherapy and Radical Cystectomy Clin. Cancer Res., December 1, 2000; 6(12): 4866 - 4873. [Abstract] [Full Text]
|
|
|
|
|
|
I. Feoktistov, A. E. Goldstein, S. Ryzhov, D. Zeng, L. Belardinelli, T. Voyno-Yasenetskaya, and I. Biaggioni Differential Expression of Adenosine Receptors in Human Endothelial Cells: Role of A2B Receptors in Angiogenic Factor Regulation Circ. Res., March 22, 2002; 90(5): 531 - 538. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
30% reduction in the mRNA expression of IL-8 and MMP-9
relative to that of either parental 253J B-V or 253J B-V(Neo). This is
one representative experiment of three.