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Cancer Biology, Immunology, Cytokines |
Departments of Cancer Biology [K. I., B. Y. E., J. W. S., S. J. K., S. Y., M. B-E., R. R., C. A. P., C. P. N. D.] and Urology [P. P., J. W. S., M. D. B., C. A. P., C. P. N. D.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Metastasis is a highly selective process involving multiple tumor-host interactions (7, 8, 9, 10, 11) . A crucial step in metastasis is vascularization in and around the tumor (12 , 13) . The balance between stimulatory and inhibitory factors released by the tumor and the microenvironment regulates this process of angiogenesis (14, 15, 16) . Human prostate cancer produces a number of proangiogenic factors, including VEGF3 (17 , 18) , bFGF, (19 , 20) , and IL-8 (18 , 21 , 22) . MVD, a pathological surrogate for angiogenesis, correlates with stage and prognosis for patients with prostate cancer (23) .
IL-8 was originally identified as a leukocyte chemoattractant (24 , 25) but is now also known to be an autocrine growth factor for malignant melanoma (26) and keratinocytes (27) . In addition, IL-8 displays mitogenic and morphogenic activity for endothelial cells (28) and regulates angiogenesis in lung cancer (29 , 30) and melanoma (31 , 32) . IL-8 is expressed by prostate cancer (18 , 21 , 22) , and this expression correlates with metastatic potential (21) . Moreover, Moore et al. (33) reported recently that neutralizing antibodies to IL-8 inhibited the angiogenic activity of PC-3 prostate cancer homogenates and reduced tumorigenicity in vivo, implicating IL-8 as an important modulator of prostate cancer growth.
Therefore, in the present study we forced the expression of IL-8 by human prostate cancer cells to determine whether IL-8 is associated with angiogenesis in vivo and to confirm the relationship between IL-8 expression and the subsequent tumorigenicity of human prostate cancer growing within the prostate of athymic nude mice. Overexpression of MMP-2 and MMP-9, which are regulated by transforming growth factor-ß, correlated with progression and poor survival of patients with prostate cancer (34, 35, 36, 37, 38) . Because IL-8 regulated the expression of MMP-2 by human melanoma (32) , we determined whether IL-8 also regulated MMP-9 expression by human prostate cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection and Selection of PC-3P and PC-3M-LN4 Cells Expressing
IL-8.
Tumor cells were plated onto 100-mm dishes at a density of 1 x
106/dish. The monolayers (60–70% confluent)
were transfected with pcDNA3/sense IL-8, pcDNA3/antisense IL-8 [a gift
from Dr. K. Matsushima (24)
], or control pcDNA3/neo
plasmids 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 modified complete MEM (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 of the lines were maintained in
CMEM/G418 and frozen after one to three in vitro passages.
To avoid clonal variations, six positive clones were then pooled for
the in vitro and in vivo studies.
The less tumorigenic and metastatic PC-3P cells and the highly tumorigenic, highly metastatic PC-3M-LN4 cells were transfected with pcDNA3/sense IL-8 or pcDNA3/antisense IL-8, respectively, or with control pcDNA3/neo. Individual G418-resistant (500–1000 µg/ml) colonies were established as separate adherent cultures. We selected pooled sense IL-8 transfected PC-3P cells [PC-3P(IL-8)], the highest IL-8-expressing clone [PC-3P(IL-8 High)], and the lowest IL-8-expressing clone [PC-3P(IL-8 Low)], and we selected pooled antisense IL-8 transfected PC-3LN4 cells [PC-3M-LN4(AS IL-8)], the highest IL-8 expressing clone [PC-3M-LN4(AS IL-8 High)], and the lowest IL-8 expressing clone [PC-3M-LN4(AS IL-8 Low)], according to 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, San Diego, CA). The mRNA was electrophoresed
onto 1% denatured formaldehyde agarose gel, electrotransferred to
Genescreen nylon membrane (DuPont, Boston, MA), and cross-linked with a
UV Stratalinker 1800 (Stratagene) at 120,000
mJ/cm2. Filters were washed twice at 65°C with
30 mM NaCl/3 mM sodium citrate-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) a 0.5-kb EcoRI cDNA
fragment corresponding to human IL-8 (a gift of Dr. K. Matsushima,
Kanazawa, Japan; Ref. 24
); (b) a 1.4-kb cDNA
fragment of bovine bFGF (41)
; (c) a 204-kb
fragment of human VEGF cDNA inserted in a pGEM-based construct (a gift
Dr. B. Berse, Harvard Medical School, Boston, MA; Ref.
42
); (d) a 1.0-kb cDNA fragment corresponding
to human MMP-9, (21)
; and (e) a 1.28-kb
fragment from pR GAPDH cut with PstI (43)
. 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]dCTP
(Amersham Corp., Arlington Heights, IL; Ref. 44
). The
steady-state expression of IL-8, bFGF, VEGF, and MMP-9 mRNA transcripts
was quantified by densitometry of autoradiographs using the Image Quant
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.
ELISA for IL-8, bFGF, and VEGF.
Viable cells (5 x 103) 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 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 absorbance comparison to the
standard curve. Results were expressed as numbers of cells
(45)
.
Growth Curve.
Viable cells (1 x 103) were seeded in a
96-well plate. Conditioned medium was removed after 24 h, and the
cells were washed with 200 µl of HBSS. Either 200 µl of fresh CRPMI
medium or CMEM/G418 conditioned medium were added. Every 24 h, the
numbers of viable cells in each cell line were determined by absorbance
comparison. The doubling time of each cell line was determined by
plotting the absorbance on a semilogarithmic axis versus
time (Cricket Software, Malvern, PA). The doubling times of the PC-3P
sense IL-8 transfectants (IL-8, 40.1 h; IL-8 Low, 38.1 h;
IL-8 High, 41.2 h) were similar to those of PC-3P (38.6 h) and
PC-3P(Neo) (39.1 h). The doubling times of the PC-3M-LN4 antisense IL-8
transfectants (AS IL-8, 22.0 h; AS IL-8 Low, 21.5 h; AS IL-8 High,
21.7 h) were similar to those of PC-3M-LN4 (22.0 h) and
PC-3M-LN4(Neo) (22.6 h).
Collagenase Activity.
To determine collagenase activity, electrophoresis of serum-free
conditioned medium was performed as described previously
(46)
. Cells (5 x 103) were
seeded in six-well plates and grown to 60–70% confluence. The cells
were washed with HBSS and grown for 24 h in serum-free medium. The
supernatant fluid was collected to determine collagenase activity, and
the remaining cells were counted to confirm the cell number. Collected
samples were centrifuged to concentrate using MICROCON
microconcentrators (Amicon, Inc., Beverly, MA.). Thirty µl in each
sample with 10 µl loading buffer (10% SDS) were electrophoresed on
20% SDS-polyacrylamide gels containing 1 mg/ml gelatin. After
electrophoresis, gels were washed in 2.5% Triton X-100 to remove SDS
and allow proteins to renature. Then gels were immersed in incubation
buffer containing 1% Triton X-100, 50 mM Tris (pH 7.5), 5
mM CaCl2, and 1 µM
ZnCl2 for 24 h at 37°C. The zymograms were
stained with 0.1% (w/v) Coomassie Blue R-250 (Sigma) and destained in
40% methanol-10% acetic acid. Identification of a transparent band
at Mr 92,000 on the Coomassie
blue background of the slab gel was considered positive for the
presence of the enzymatic activity. The collagenase activity was
quantified using densitometry of Image Quant software program
(Molecular Dynamics, Sunnyvale, CA).
To determine whether the increase in MMP-9 activity is mediated by IL-8, we incubated parental PC-3P cells in the presence of different doses (0–20 µg/ml) of human rIL-8, and the activity of MMP-9 was determined. We then determined the increased activity of MMP-9 by rIL-8 was inhibited by neutralization by using an anti-IL-8 antibody (100 µg/ml), with a nonspecific IgG (100 µg/ml) as a control.
PCR Analysis.
RT-PCR analysis was performed as described previously
(47)
. Briefly, total cellular RNA (1 mg) extracted from
various cell lines was transcribed into cDNA using downstreaming
primers IL-8 receptors type A and B, respectively (Reverse
Transcription System;, Promega). 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),
and 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: for IL-8 receptor type A, sense
5-AGT TCT TGG CAC GTC ATC G-3'and antisense 5'-CTT GGA GGT ACC TCA ACA
GC-3'; and for IL-8 receptor type B, sense 5'-ACA TTC CTG TGC AAG GTG
G-3' and antisense 5'-CAG GGT GAA TCC GTA GCA GA-3'.
Invasion Assay 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 mmol/l), centrifuged, resuspended in 0.1%
BSA-DMEM, and placed in the upper compartment of the Boyden chamber.
Fibroblast-conditioned medium in the lower compartment served as a
chemoattractant. 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, McGraw Park, IL) and quantified with a cooled CCD
Optotronics Tec 470 camera (Optotronics Engineering, Goletha, CA)
linked to a computer and digital printer (Sony Corporation, 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 (48)
.
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 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 PC-3P, PC-3M-LN4, Neo, sense, and antisense IL-8 transfected
cells (60–70% confluent) were prepared for injection as described
previously (39
, 49)
. Mice were anesthetized with
methoxyflurane. For orthotopic implantation, a lower midline incision
was made, and viable tumor cells (2 x
106/40 µl) in HBSS were implanted into the
dorsal prostate lobes using a 30-gauge needle with a 1-ml disposable
syringe and a calibrated push button-controlled dispensing device
(Hamilton Syringe Company, Reno, NV). Formation of a bulla indicated a
satisfactory injection. The prostate was returned to the abdominal
cavity, and the abdominal wall was closed with a single layer of metal
clips. Mice were killed 6 weeks after implantation of tumor cells. The
primary tumors were removed and weighed, and the presence of metastases
(in the lymph nodes) was determined grossly and microscopically. The
prostates 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.
ISH Analysis.
Specific antisense 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% guanosine
cytosine (GC) content (21)
; bFGF (CGG GAA GGC GCC GCT GCC
GCC), 85.7% GC content (41)
; VEGF/VPF (TGG TGA TGT TGG
ACT CTT CAG TGG GCU), 57.7% GC content (42)
; and MMP-9 (CCG GTC CAC
CTC GCT GGC GCT CCG GU), 80.0% GC content (21)
. 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
(50)
. 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
(hyperbiotinylated) at the 3' end via direct coupling with the use of
standard phosphoramidite chemical methods (Research Genetics,
Huntsville, AL). The lyophilized probes were reconstituted to a stock
solution at 1 µg/µl in 10 mmol/l Tris (pH 7.6) and 1 mmol/l EDTA.
Immediately before use, the stock solution was diluted with probe
dilution (Research Genetics, Huntsville, AL).
In situ mRNA hybridization was performed as described previously with minor modifications, (51 , 52) using the Microprobe Manual Staining System (Fisher Scientific, Pittsburgh, PA; Ref. 53 ). Tissue sections (4 µm) of formalin-fixed, paraffin-embedded specimens were mounted on silane-treated ProbeOn slides (Fisher Scientific; Refs. 51 and 52 ). 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 sodium saline chloride (Research Genetics) 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. Additional incubation with fresh chromogen substrate was performed if necessary to enhance a weak reaction in this assay; a red staining indicated a positive reaction. Control for endogenous alkaline phosphatase included treatment of the sample in the absence of the biotinylated probe and the use of 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., Tokyo, Japan). The images were
analyzed using the Optimas image analysis software (version 4.10;
Bioscan, 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 each field 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 cells for each cell line compared with the control, which was set to
100 (45)
.
IHC.
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, antigen retrieval was performed with
pepsin for 12 min, and 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 the
appropriate dilution (1:100) of rat monoclonal anti-CD31 antibody
(PharMingen, San Diego, CA; Ref. 54
), 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, San Diego, CA).
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 again washed three times with PBS.
The slides were mounted with Universal Mount mounting medium (Research
Genetics).
Quantification of Microvessel Density.
Microvessel density was determined by light microscopy after
immunostaining frozen sections with anti-CD31 antibodies as described
by Weidner et al. (55)
. 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 CCD Optotronics Tec 470 camera (Optotronics
Engineering, Goletha, CA) 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
quantitated in each sample by an image analyzer using the Optimas
software program (Bioscan). Three different areas in each sample were
quantified to yield an average measurement. The results were presented
as the number of cells for each cell line compared with the control,
which was set to 100 (45)
.
MMP-9 mRNA Half-Life Studies.
To determine the effect of IL-8 on MMP-9 mRNA stability, PC-3P,
PC-3P(Neo), and PC-3P(IL-8) cells and PC-3M-LN4, PC-3M-LN4(Neo), and
PC-3M-LN4(AS IL-8) cells were incubated for 24 h. Further
transcription in the 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 at 0,
1, 2, and 4 h after the addition of ActD, and MMP-9 mRNA
expression was determined by Northern blot analysis. 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).
CAT Assay.
Using the FuGENE 6 protocol (Boehringer Mannheim Corp.), we transfected
with the basic CAT expression vector with no promoter/enhancer
sequences (pCAT-basic) or a control plasmid with SV40 promoter and
enhancer (pCAT-control; Promega Corp., Madison, WI) into PC-3P cells,
sense-transfected PC-3P cells, PC-3M-LN4 cells, antisense-transfected
PC-3M-LN4 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 in a six-well tissue culture dish
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 (56)
, as Hudson et al.
(57)
described previously. Each assay was repeated twice;
there was <10% variation in transfection efficiency. The CAT assay
was quantified by densitometry of autoradiographs with the use of the
ImageQuant software program (Molecular Dynamics, Sunnyvale, CA) and was
evaluated as the ratio of acetylated species to all species.
Statistical Analysis.
The Mann-Whitney U test analyzed the statistical differences
in vessel counts and staining intensity for IL-8, bFGF, VEGF, and MMP-9
of prostate tumors. The incidences of tumor and metastasis were
statistically analyzed by 
2 test. A value of
P < 0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and that by PC-3M-LN4,
PC-3M-LN4(AS IL-8), PC-3M-LN4(AS IL-8 Low), PC-3M-LN4(AS IL-8 High),
and PC-3M-LN4(Neo) is shown in Fig. 1B
. The level of
expression is shown as the ratio of mRNA expression by the
transfectants to that by the corresponding parental and Neo
transfectant cell lines (which in both cases was equivalent for all
three factors). IL-8 mRNA expression levels were increased 10.4- and
15.1-fold higher in PC-3P(IL-8) and PC-3P(IL-8 High), respectively,
than in either PC-3P or PC-3P(Neo), whereas there was no change in the
mRNA expression of bFGF or VEGF. The mRNA expression levels of IL-8 in
PC-3M-LN4(AS IL-8) and in PC-3M-LN4(AS IL-8 Low) were only about
one-fifth and one-tenth of those for either PC-3M-LN4 or
PC-3M-LN4(Neo), respectively, whereas there was no change in the mRNA
expression of bFGF or VEGF. IL-8, bFGF, and VEGF protein production by
PC-3P, PC-3M-LN4, 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.
IL-8 expression levels were 3.0- and 4.0-fold higher in the PC-3P(IL-8)
and PC-3P(IL-8 High) cells, respectively, than in the PC-3P parental
cell line. PC-3M-LN4(AS IL-8) and PC-3M-LN4(AS IL-8 Low) cell lines
were only one-quarter and one-tenth of those in the parental PC-3M-LN4
cells, respectively. bFGF and VEGF protein expression levels were
unchanged in all cell lines after transfection.
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shows that MMP-9 mRNA expression levels were 3.0-
and 6.0-fold higher in PC-3P(IL-8) and PC-3P(IL-8 High) cells,
respectively, compared with their controls, but the values for both
PC-3M-LN4(AS IL-8) and PC-3M-LN4(AS IL-8 Low) cells were only one-third
of that of their controls. These results demonstrate that IL-8
regulates MMP-9 mRNA expression by the PC-3P and PC-3M-LN4 human
prostate cancer cells. Our results are consistent with reports that
IL-8 regulates MMP expression by malignant melanoma (31
, 32)
.
Collagenase Activity.
To demonstrate that MMP-9 expressed by the transfected cells is
biologically active, collagenase activity of the transfected cells was
determined by zymography after normalizing the volume of supernatant
for cell number (Fig. 3)
. By
densitometry, the collagenase activity of PC-3P(IL-8) and PC-3P(IL-8
High) cells was increased 6.0- and 7.0-fold, compared with either PC-3P
or PC-3P(Neo) (Fig. 3A)
, respectively, whereas that of
PC-3M-LN4(AS IL-8) and PC-3M-LN4(AS IL-8 Low) was decreased 2.5- and
5.0-fold compared with either PC-3M-LN4 or PC-3M-LN4(Neo), respectively
(Fig. 3B)
.
|
. To that end, parental
PC-3P 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)
.
|
, as well as PC-3M-LN4,
PC-3M-LN4(Neo), and antisense IL-8 transfectants (AS IL-8, AS IL-8 Low,
and AS IL-8 High; Fig. 5B)
, express mRNA for both IL-8
receptors.
|
. Invasion by PC-3M-LN4(AS
IL-8) and PC-3M-LN4(AS IL-8 Low) was 80 and 85% lower, compared with
invasion by PC-3M-LN4 or PC-3M-LN4(Neo) (P < 0.005),
respectively (Fig. 6B)
.
|
compared with either PC-3P
or PC-3P(Neo), respectively, and decreased 2.5- and 10.0-fold by
PC-3M-LN4(AS IL-8) and PC-3M-LN4(AS IL-8 Low) (Fig. 7B)
compared with either PC-3M-LN4 or PC-3M-LN4(Neo), respectively. CAT
activity driven by the SV40 promoter was the same in both cell
populations and served as an additional internal control for
transfection efficiency. Next, the stability of MMP-9 mRNA was
investigated by examining its half-life. The half-life of MMP-9 mRNA of
PC-3P(IL-8) was similar to that of PC-3P or PC-3P(Neo), and that of
PC-3M-LN4(AS IL-8) was similar to that of PC-3M-LN4 or PC-3M-LN4(Neo)
(data not shown).
|
. The PC-3P(IL-8) and PC-3P(IL-8 High)
tumors were larger than the PC-3P and PC-3P(Neo) tumors [mean weight
(range; P): 1270 mg (258–1850 mg; P <
0.005) and 1975 mg (355–2915 mg; P < 0.005)
versus 55 mg (24–480 mg) and 120 mg (27–443 mg)].
Moreover, the incidences of spontaneous lymph node metastasis, as well
as tumor burden within the metastatic lymph nodes, were significantly
greater for the PC-3P(IL-8) and PC-3P(IL-8 High) cell lines than for
the PC-3P and PC-3P(Neo) [incidences: 8 of 8, 9 of 9, 4 of 7, 4 of 9,
respectively (P < 0.05); mean lymph node weight
(range): 93 mg (36–124 mg), 131 mg (55–188 mg), 20 mg (16–28 mg),
and 24 mg (15–32 mg), respectively; P < 0.005].
Conversely, the tumorigenicity of PC-3M-LN4 was significantly inhibited
when IL-8 expression was reduced by antisense IL-8 transfection. Only
two of eight mice implanted with PC-3M-LN4(AS IL-8) (tumor weights, 33
and 36 mg) and PC-3M-LN4(AS IL-8 Low) (tumor weights, 31 and 39 mg)
developed tumors at 6 weeks (P < 0.01) compared with
the mice implanted with PC-3M-LN4 and PC-3M-LN4(Neo). These tumors were
not apparent histologically but grew out as tissue explants in culture.
Tumorigenicity was also inhibited in PC-3M-LN4(AS IL-8 High), but this
difference did not reach statistical significance. There was a
significant reduction in spontaneous lymph node metastases at 6 weeks
in mice implanted with PC-3M-LN4(AS IL-8), PC-3M-LN4(AS IL-8 Low), or
PC-3M-LN4(AS IL-8 High) (no mice developed metastasis) compared with
mice implanted either PC-3M-LN4 or PC-3M-LN4(Neo) (all mice developed
metastasis; P < 0.0005). Therefore, IL-8 expression by
PC-3P and PC-3M-LN4 regulates both tumorigenicity and metastasis in
androgen-independent prostate cancer.
|
; Fig. 8
) and IHC (Table 2
; Fig. 9
), respectively.
The mRNA and protein expressions of IL-8 and MMP-9 were increased 2.5-
and 3.0-fold in the PC-3P(IL-8) and PC-3P(IL-8 High) tumors,
respectively, relative to either PC-3P or PC-3P(Neo). The PC-3M-LN4(AS
IL-8 High) tumors showed a 42% reduction in the mRNA and protein
expression of IL-8 and a 33% reduction in the mRNA and protein
expression of MMP-9 relative to either PC-3M-LN4 or PC-3M-LN4(Neo)
tumors. There was no change in the mRNA and protein expression of bFGF
or VEGF in the IL-8-transfected tumors.
|
|
|
; Fig. 9
). The numbers of
CD31+ microvessels counted per x200 field were
40 ± 9 and 45 ± 6 in PC-3P and PC-3P(Neo), respectively,
compared with 80 ± 13 and 91 ± 18 in PC-3P(IL-8) and
PC-3P(IL-8 High), respectively (P < 0.005).
Conversely, antisense IL-8 transfection of PC-3M-LN4 significantly
decreased MVD from 100 ± 20 and 104 ± 23 in the PC-3M-LN4
and PC-3M-LN4(Neo) tumors, respectively, to 47 ± 14 in the
PC-3M-LN4(AS IL-8 High) tumors (P < 0.005; Table 2
;
Fig. 9
). Because the PC-3M-LN4(IL-8) and PC-3M-LN4(AS IL-8 Low) cells
grew only as explants in tissue culture, we could not evaluate MVD.
These studies indicate that tumor-induced neovascularization correlates
directly with IL-8 expression, tumorigenicity, and metastasis. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We enforced IL-8 expression by transfecting the poorly tumorigenic and poorly metastatic human prostate cancer cell line PC-3P (which expresses relatively low levels of IL-8) with the sense IL-8 construct and were able to establish several cell lines that overexpress IL-8 relative to the original PC-3P cell line. These sense IL-8-transfected cells demonstrated enhanced tumor growth and metastasis compared with the PC-3P or PC-3P(Neo) cells and demonstrated enhanced tumor-induced neovascularization, growth within the prostate, and spontaneous metastasis to the lymph nodes. Conversely, after antisense IL-8 transfection, we were able to reduce IL-8 expression by the highly tumorigenic and metastatic PC-3M-LN4 cell line (which expresses relatively high levels of IL-8) and to inhibit tumor-induced neovascularization, growth within the prostate, and metastasis. 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. Because IL-8 transfection did not affect in vitro proliferation of PC-3P or PC-3M-LN4, the effects on growth and metastasis are independent of proliferation, although the cells do have both type A (CXCR1; Refs. 59 and 60 ) and type B (CXCR2; Refs. 59 and 60 ) of the IL-8 receptors. Therefore, our results provide evidence for the involvement of IL-8 in the induction of in vivo angiogenesis and in the subsequent growth and metastasis of prostate cancer. 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 (61, 62, 63) . However, because of the differences in tumor size between control and IL-8 sense and antisense transfectants, the difference in tumor-induced angiogenesis and in metastatic potential may reflect tumor size.
The metastatic potential of prostate cancer depends upon the expression of several metastasis-related genes, such as IL-8, that regulate endothelial cell proliferation and capillary morphogenesis (28) , and other genes, such as MMP-9, that regulate the degradation of the extracellular matrix (35 , 64) . The local production of MMP-9 or other proteases, such as plasminogen activator, by prostate cancer cells or stroma facilitates the local degradation of the extracellular matrix and results in tumor invasion and subsequent metastasis (35 , 64, 65, 66) . 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 PC-3M-LN4 expresses high levels of MMP-9 compared with the poorly metastatic PC-3P cell line. Recently, Luca et al. (32) reported that IL-8 regulates MMP-2 activity by malignant melanoma cells. They transfected the melanoma cell line SB-2 with the sense IL-8 transcript and up-regulated MMP-2 expression and collagenase activity. They considered this up-regulation of collagenase activity to be an important mechanism that explained the associated increase in metastatic ability demonstrated by the sense-transfected SB-2 cells. Similarly, we found that the activity of both MMP-9 by human prostate cancer cells directly correlated with their expression of IL-8. Moreover, when we altered the expression of IL-8 by sense or antisense transfection, we observed a corresponding change in MMP-9 expression and activity both in vitro and in vivo. The MMP-9 induced by sense transfection was biologically active, because it increased collagenase activity and increased cellular invasion through Matrigel. When MMP-9 activity was reduced after antisense transfection, both collagenase activity and invasion through Matrigel decreased. The altered local growth of the antisense-transfected tumors may reflect a relative growth inhibition secondary to the inability to induce a robust microcirculation, whereas the loss of metastatic potential may be attributable to both a decrease in the tumor-induced neovascularization by IL-8 and a reduction in invasion attributable to the reduction in MMP-9 activity. Conversely, increased IL-8 expression by the sense IL-8-transfected prostate cancer cells may explain their enhanced tumorigenicity, whereas both increased IL-8 and MMP expression may contribute to their increased metastatic potential. Because these experiments were conducted in athymic nude mice, they do not address the well-established role of IL-8 in tumor immunity (25) .
MMP-9 expression is regulated by both transcriptional and posttranscriptional events. Whereas bFGF and VEGF regulate the transcription of MMP-9 through activation of Ets-1 and Ets-2 binding sites in the promoter (67 , 68) , transforming growth factor-ß1 up-regulates MMP-9 by increasing mRNA stability (37) . IL-8 probably regulates MMP-9 expression at the level of transcription. 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 or antisense IL-8. However, CAT activity driven by the MMP-9 promoter was up-regulated in IL-8 sense transfectants and down-regulated after antisense transfection. bFGF regulates MMP-9 expression in human bladder cancer. Because bFGF levels were not affected by IL-8 transfection, the regulation of MMP-9 transcription in PC-3P and PC-3M-LN4 cells is independent of bFGF and likely regulated by IL-8. These results are in keeping with the report of Luca et al. (32) , who found that IL-8 regulated MMP-2 gene transcription.
In summary, our present study demonstrates that IL-8 regulates angiogenesis, tumorigenesis, MMP-9 expression, and metastasis by androgen-independent human prostate cancer. This effect may be mediated, in part, by the regulation of the expression and activity of MMP-9.
|
FOOTNOTES |
|---|
1 Supported in part by NIH Grants CA67914,
CA56973, and Core Grant CA-16672, a grant from the Department of
Defense, and a grant from the Robert Wood Johnson Foundation. 
2 To whom requests for reprints should be
addressed, Department of Urology, Box 173, The University of Texas
M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX
77030. Phone: (713) 792-3250; Fax: (713) 794-4824; E-mail: cdinney{at}mdanerson.org
3 The abbreviations used are: VEGF, vascular
endothelial cell growth factor; bFGF, basic fibroblast growth factor;
IL, interleukin; rIL, recombinant IL; MMP, matrix metalloproteinase;
MVD, microvessel density; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; CMEM, complete Eagle’s minimum essential medium; ISH,
in situ hybridization; IHC, immunohistochemical
staining; ActD, actinomycin D; CAT, chloramphenicol acetyltransferase;
RT-PCR, reverse transcription-PCR.
Received 12/ 8/99; revised 2/22/00; accepted 2/23/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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D. Giri and M. Ittmann Interleukin-8 Is a Paracrine Inducer of Fibroblast Growth Factor 2, a Stromal and Epithelial Growth Factor in Benign Prostatic Hyperplasia Am. J. Pathol., July 1, 2001; 159(1): 139 - 147. [Abstract] [Full Text] [PDF]
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G. Opdenakker, P. E. Van den Steen, B. Dubois, I. Nelissen, E. Van Coillie, S. Masure, P. Proost, and J. Van Damme Gelatinase B functions as regulator and effector in leukocyte biology J. Leukoc. Biol., June 1, 2001; 69(6): 851 - 859. [Abstract] [Full Text] [PDF]
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 J. I. Izawa and C. P.N. Dinney The role of angiogenesis in prostate and other urologic cancers: a review Can. Med. Assoc. J., March 1, 2001; 164(5): 662 - 670. [Abstract] [Full Text] [PDF]
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A. Gujral, D. W. Burton, R. Terkeltaub, and L. J. Deftos Parathyroid Hormone-related Protein Induces Interleukin 8 Production by Prostate Cancer Cells via a Novel Intracrine Mechanism Not Mediated by its Classical Nuclear Localization Sequence Cancer Res., March 1, 2001; 61(5): 2282 - 2288. [Abstract] [Full Text]
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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]
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