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Regular Articles |
From the Departments of Internal Medicine*
and Pathology
and the Undergraduate Research
Opportunities Program,
University of Michigan
Medical Center, Ann Arbor, Michigan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, to mediate
tumorigenicity. Neutralizing antisera to GRO- but not IL-8 reduced
tumor growth in vivo and reduced the angiogenic activity
in tumor homogenates. Thus, prostate cancer cell lines can use
distinct CXC chemokines to mediate their tumorigenicity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CXC chemokines are cytokines whose
expression can be induced by inflammatory stimuli in all nucleated
cells in the body,4-9
and these molecules have recently
been shown to modulate angiogenesis. Based on the presence or absence
of the `ELR' motif (Glu-Leu-Arg) preceding the first conserved
cysteine amino acid in the NH2 terminus, this
family can be divided into two groups. The first group of CXC
chemokines contains the ELR motif and is angiogenic. These include
interleukin-8 (IL-8), epithelial-neutrophil activating protein
(ENA-78), growth-related genes (GRO-, GRO-ß, and GRO-
),
granulocyte chemotactic protein (GCP-2), and
NH2-terminal truncations of platelet basic
protein (connective tissue activating protein (CTAP-III),
ß-thromboglobulin, and neutrophil activating protein
(NAP-2)).10,11
The second group of CXC chemokines, which
lack the ELR motif, include interferon--inducible protein (IP-10),
monokine induced by -interferon (MIG), platelet factor 4 (PF4), and
stromal derived factor (SDF-1).10,11
Interestingly, these
ELR-negative CXC chemokines, in general, have been shown to antagonize
the angiogenic activities of the ELR-positive CXC chemokines as well as
basic fibroblast growth factor (bFGF) and vascular endothelial growth
factor (VEGF).10
IL-8, an angiogenic CXC chemokine, is significantly elevated in
non-small cell lung cancer (NSCLC).12,13
In addition, IL-8
is a significant angiogenic factor contributing to overall
tumor-derived angiogenic activity in NSCLC.13
Furthermore,
human gastric carcinomas express high levels of IL-8.14
The levels of IL-8 in these neoplasms strongly correlate with their
vascularity, suggesting that IL-8 produced by gastric tumor cells may
regulate neovascularization of gastric carcinoma. Survival of nude mice
injected with ovarian carcinomas have been shown to be inversely
correlated with the production of IL-8 by the ovarian
cancers.15
Similarly, recent reports indicate that
significant levels of IL-8 are present immunohistochemically in
prostate cancer specimens but not in benign prostate hyperplasia or
normal prostate cells.16
In a similar manner, GRO-
over-expression has been reported in melanoma lesions.17
These neoplasms show the expression of the putative receptor for
GRO-, CXCR2, on both the melanoma cells and tumor-infiltrating
endothelial cells. Stable transfection of the human GRO- gene into
the murine melan-a-melanocyte line resulted in significantly enhanced
tumorigenicity that, in part, was related to their enhanced angiogenic
phenotype.17
Although these studies certainly support the
hypothesis that CXC chemokines can mediate tumor-associated
angiogenesis, it remains to be elucidated whether their angiogenic
behavior is universal to all solid tumors or whether successful
anti-tumor therapies can be established by antagonizing the effects of
particular angiogenic CXC chemokines. For these reasons, we have
analyzed the role CXC chemokines play in mediating tumorigenesis in
association with prostate cancer.
We demonstrate here that the human prostate cancer cell lines, PC-3 and
Du145, constitutively over-express angiogenic CXC chemokines and that
each cell line uses unique CXC chemokines to mediate their tumor
growth. Using a human prostate cancer/SCID mouse chimera system, we
demonstrate that IL-8 is a major positive regulator of
neovascularization and tumorigenesis associated with the growth of PC-3
tumors. In contrast, the Du145 cell line primarily uses a different
ELR-CXC chemokine, GRO-, in a paracrine manner to support an
angiogenic environment during tumorigenesis. These data further support
the hypothesis that individual CXC chemokines can regulate
tumor-associated angiogenesis and demonstrate that anti-CXC chemokine
therapy directed at one particular chemokine may not be entirely
beneficial even within the same tumor type.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The prostate cancer cell lines were maintained in sterile 150 mm2 tissue culture flasks in appropriate media as defined by American Type Culture Collection (Rockville, MD). PC-3 cells grow in Ham's F12K media, Du145 cells in EMEM, and LNCaP cells in RPMI 1640 (Whitaker Biomedical Products, Whitaker, CA) supplemented with 1 mmol/L glutamine, 25 mmol/L HEPES buffer, 100 units/ml penicillin, 100 ng/ml streptomycin (Whitaker Biomedical Products), and 10% fetal calf serum (Harlan, Indianapolis, IN). Cells were cultured and passaged at 37°C in 5% CO2.
In Vitro Enzyme-Linked Immunosorbent Assay (ELISA) Characterization
Approximately 2 x 106 prostate cancer cells were plated in 60-mm2 culture plates in appropriate media. The next day, the media was replaced with fresh growth media, and cell-free supernatants were collected from replicate plates at 24-, 48-, 72-, and 96-hour intervals. Total antigenic levels of CXC chemokines in culture supernatants were assessed by specific ELISAs as previously described.18,19
Neutralizing Antibodies
The goat anti-human IL-8 antibody was generated against an
N-terminal IL-8 peptide by a modification of the previously described
protocol to generate rabbit anti-human IL-8 antibodies
(Abs).13,20,21
The rabbit anti-human GRO-
neutralizing antisera was generated as previously described by
injecting female New Zealand White rabbits in multiple intradermal
sites with recombinant GRO- in complete Freund's adjuvant with
subsequent boosts given in incomplete adjuvant. The anti-GRO- and
anti-IL-8 antisera each neutralize 30 ng of their respective chemokine
at a dilution of 1:1000 without cross-reactivity. These Abs have each
been shown to be specific for their respective chemokines and do not
cross-react with the murine homologues of these chemokines.
In Vitro Proliferation Assays
5000 PC-3 or Du145 cells were cultured in 96-well flat-bottomed
tissue culture plates in their respective media. To the media was added
1, 10, or 100 ng/ml recombinant IL-8 or GRO or vehicle control. In
addition, PC-3 and Du145 cells were cultured in the presence of 1:10 or
1:100 dilutions of the neutralizing anti-IL-8 or anti-GRO antisera
as well as the same dilutions of a normal preimmune serum control.
Final volumes were 200 µl per well. Cells were cultured under these
conditions for 24 or 48 hours before 1 µCi of
3-H thymidine was added for 16 hours. Plates were
harvested on a Brandel cell harvester and filters were counted in a
Beckman LS-1801 ß counter. Data in Table 1
represent at least six replicate wells
plated for each condition and each time point.
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Male SCID mice between ages 4 to 6 weeks were used. A cohort group of SCID mice were injected with 1 x 106 prostate cancer cells in 100 µl of serum free media into each flank region (bilateral). All SCID mice were monitored daily for evidence of illness. Measurement of tumor size by a Thorpe engineer's caliper (Biomedical Research Instruments, Rockville, MD) was done on a weekly basis. Tumor volume was calculated using the formula, volume = (d1 x d2 x d3) x 0.5236, in which dn represents the three orthogonal diameter measurements. A portion of the tumor was fixed in 4% paraformaldehyde for histological analysis and immunohistochemistry. In the experiments using neutralizing antibodies in vivo, prostate cancer bearing SCID mice received intraperitoneal (i.p.) injections of 0.5 ml of either neutralizing antisera or appropriate control (preimmune) rabbit or goat serum at the time of cell innoculum and every 48 hours thereafter for the duration of the study.
In Vivo Analysis of CXC Chemokines
Two random punch biopsies of each tumor were taken and immediately snap-frozen in liquid nitrogen. Tumor tissue was further processed by homogenization and sonication in an anti-protease "cocktail" of 2 mmol/L phenyl methyl sulfonyl fluoride, and 1 µg/ml each of antipain, aprotinin, leupeptin, and pepstatin A. Samples were then analyzed for antigenic CXC chemokine levels by specific ELISA and normalized to total protein (as measured by the BCA assay, Pierce Chemicals, Rockford, IL) as previously described.18,19
Corneal Micropocket Model of Angiogenesis
In vivo angiogenic activity of human tumors was assayed in the avascular cornea of Long Evans rat eyes as previously described.10,12,13,19 Briefly, equal volumes of lyophilized tumor specimens normalized to total protein were combined with sterile Hydron (Interferon Sciences Inc.) casting solution. Five-µl aliquots were pipetted onto the flat surface of an inverted sterile polypropylene specimen container and were polymerized overnight under UV light in a laminar flow hood. Before implantation, pellets were rehydrated in normal saline. Animals were given i.p. ketamine (150 mg/kg) and atropine (250 µg/kg) for anesthesia. Rat corneas were anesthetized with 0.5% proparracaine hydrochloride ophthalmic solution followed by implantation of the Hydron pellet into an intracorneal pocket (1 to 2 mm from the limbus). Six days after implantation, animals received heparin (1000 U) and ketamine (150 mg/kg) i.p. followed by perfusion with 10 ml of colloidal carbon via the left ventricle. Corneas were harvested and photographed. Positive neovascularization responses were defined as sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant. Negative responses were defined as either no growth or only an occasional sprout or hairpin loop displaying no evidence of sustained growth.
Quantification of Vessel Density
Endothelial cells in tumor specimens were enumerated by FACS analysis of PECAM (CD31). Tumor samples were digested to single cell suspensions via mincing followed by enzymatic digestion and agitation (2 hours, 37°C) in Dispase (Collaborative Biomedical Products, Bedford, MA). Cells were then stained and analyzed by flow cytometry for PECAM (CD31) (PharMingen, San Diego, CA) expression using a modification as previously described.19 The vascularity index was generated by multiplying the percentage of CD31-positive cells (endothelial cells as assessed by flow cytometry) by the tumor volume.
FACS Analysis for CXCR1 and CXCR2 Expression
CXCR1 and CXCR2 receptor expression was analyzed on PC-3 and Du145 cells as well as on normal human peripheral blood neutrophils. The Abs used were from Pharmingen (San Diego, CA). The anti-CXCR1 Ab is clone 5A12, which we used fluorescein isothiocyanate conjugated. The anti-CXCR2 Ab is clone 6C6, which we used PE-conjugated. Samples were analyzed on a Becton-Dickinson FACScan.
Statistical Analysis
The animal studies involved a minimum of 12 human prostate tumors or six SCID mice at each time point or for each manipulation. Data that appeared statistically significant were compared by the Mann-Whitney U test. Data were considered significant if P values were less than 0.05. Results are presented as means ± SEM. Data were analyzed by a Macintosh IIfx computer using the software package InStat for Macintosh (GraphPad Software Co.). R values for correlations were generated using Statview 4.5 (Abacus Concepts Inc., Berkely, CA).
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Results |
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PC-3,22
Du145,23
and
LNCaP24
are human prostate cancer cell lines derived from
prostate cancer patients. PC-3 was originally derived from a prostatic
adenocarcinoma metastatic to bone. Du145 was derived from a prostatic
adenocarcinoma metastatic to brain, and LNCaP was derived from a needle
biopsy of the supraclavical lymph node of a patient with metastatic
prostate cancer. These cell lines were grown to confluence in
60-mm2
tissue culture dishes, the media was
changed, and cell-free supernatants were collected at 24, 48, 72, and
96 hours to evaluate constitutive production of angiogenic CXC
chemokines. This conditioned media was analyzed for the presence of
IL-8, ENA-78, GRO-, and GRO- by specific ELISA. Figure 1
represents the ELISA results obtained
from the 96-hour conditioned media. Both PC-3 and Du145 prostate cancer
cell lines constitutively produced significant levels of angiogenic CXC
chemokines. In contrast, LNCaP cells expressed 150- and 25-fold less
IL-8 than PC-3 and Du145 cells, respectively. In addition, LNCaP cells
expressed 82- and 14-fold less GRO- than PC-3 and Du145 cells,
respectively. Furthermore, the remainder of the angiogenic CXC
chemokines were reduced from LNCaP cells. Based on the previous
observations that these members of the CXC chemokine family are
angiogenic, we hypothesized that the PC-3 and Du145 cell lines would
display an angiogenic phenotype.
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To determine whether the over-expression of angiogenic CXC
chemokines by the PC-3 and Du145 cell lines in vitro
correlated with their tumorigenicity in vivo, 1 x
106
human prostate cancer cells were injected
into each flank of male SCID mice. These mice were then carefully
monitored for tumor growth for a period of 9 weeks. The experiments
were terminated at 9 weeks because of morbidity noted secondary to
tumor burden in the animals injected with PC-3 cells. The PC-3 and
Du145 cell lines produced easily discernible tumors by week 3, which
grew progressively over the 9 weeks of the study (Figure 2)
. In contrast, the LNCaP line showed no
discernible tumor growth beyond the original injection site. In
addition, metastatic lesions were seen histologically in the lungs of
both the PC-3 and Du145 tumor-bearing animals at week 8 (data not
shown).
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To ascertain whether angiogenic CXC chemokine expression directly
correlated with human prostate cancer tumorigenesis in vivo,
we xenografted 1 x 106
PC-3 or Du145 cells
subcutaneously into the flanks of a large cohort of male SCID mice. The
above studies suggested that the angiogenic CXC chemokines were
constitutively produced at moderate to high levels by PC-3 and Du145
cells in vitro, therefore we postulated that these
angiogenic chemokines may be acting in vivo to induce
neovascularization and support tumorigenesis. To test this hypothesis,
PC-3 or Du145 tumor-bearing mice were sacrificed weekly beginning 1
week after injection, and the level of angiogenic ELR-CXC chemokines in
the tumors was measured by ELISA. Figure 3
demonstrates the correlations between
tumor volume and angiogenic CXC chemokine production in vivo
in PC-3 tumor-bearing mice. There is a positive correlation between the
production of IL-8 and tumor volume (Figure 3A
, r =
0.723 for weeks 1 through 6). This increase in IL-8 also correlated
with an increase in tumor mass. In addition, in vivo
production of ENA-78 seemed to correlate with the exponential tumor
growth seen during weeks 6 to 8 (Figure 3C
, r =
0.973). However, there was apparently no correlation between PC-3 tumor
growth and tumor-derived production of GRO- (Figure 3B
,
r = 0.238) or GRO- (Figure 3D
, r =
-0.787).
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demonstrates a positive
correlation between tumor-derived IL-8 production and tumor volume
(r = 0.955). In addition, Figure 4B
demonstrates
a positive correlation between Du145 tumor growth and tumor-derived
GRO- (r = 0.879). As can be seen in Figures 4C and 4D
however, there is no clear evidence that ENA-78
(r = -0.478) and GRO-
(r = -0.568) are correlated with Du145
tumorigenicity.
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,
suggesting that this factor supports tumor growth through a paracrine
(angiogenic) mechanism rather than as an autocrine (mitogenic)
mechanism. Inhibition of IL-8 in Vivo Reduces PC-3 Tumorigenicity but Fails to Reduce Du145 Tumorigenicity
Having established that IL-8 was a common factor expressed by the
PC-3 and Du145 cell lines both in vitro and in
vivo, we wanted to determine whether IL-8 was directly
contributing to the tumorigenicity of these cells in vivo.
To do this, two groups of male SCID mice were injected bilaterally as
before with either 1 x 106
PC-3 or Du145
cells in the rear flanks. Starting at time of tumor cell innoculum,
half the animals in each group were given an i.p. injection of a
neutralizing polyclonal goat anti-human IL-8 antisera (0.5 ml). The
other half of the mice were given an 0.5 ml i.p. injection of normal
preimmune goat serum (NGS) as a control. The injections were repeated
every 48 hours for the duration of the study, and tumors were measured
weekly. Figure 5
demonstrates the
resulting tumor growth (as indicated by tumor volume) in the anti-IL-8-
versus NGS-treated animals. Figure 5A
demonstrates that
anti-IL-8 treatment clearly blocked tumor progression in PC-3 tumor
bearing mice. The mean tumor volume in the NGS-treated PC-3
tumor-bearing mice at week 7 was 1492 ± 211
mm3. The mean volume in the anti-IL-8 treated
PC-3 tumor-bearing mice was 736 ± 166 mm3.
This change represents a greater than 49% inhibition
(P = 0.01). Similarly, the vascularity index was
3.3-fold less in the PC-3 tumor-bearing animals treated with anti-IL-8
(23,749 ± 4640) than in the PC-3 animals treated with NGS
(77,218 ± 17,528). Furthermore, when tumor homogenates from the
NGS- and anti-IL-8-treated PC-3 tumor-bearing mice were concentrated,
normalized to total protein, embedded into a hydron pellet, and
implanted into the avascular cornea of a Long Evans rat (the rat
corneal micropocket assay; CMP), six of six corneas tested with the
NGS-treated homogenates gave positive neovascular responses (Figure 6A)
, whereas in the anti-IL-8-treated
tumor homogenates tested, five of six were negative or weakly positive,
giving an occasional loop as depicted in Figure 6B
, and one of six
corneas tested was positive.
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, nor did they show
any histological differences (data not shown). In an effort to confirm
that the dose of anti-IL-8 given was sufficient to neutralize the IL-8
being produced by the Du145 tumors in vivo, tumor
homogenates from tumor-bearing mice were processed and analyzed by
specific ELISA for the expression of the angiogenic CXC chemokines. The
production of IL-8 by these Du145 tumor cells was significantly
neutralized by the administration of the anti-IL-8 Abs. Du145 tumor
homogenates from NGS-treated animals produced 0.751 ± 0.281 ng/ml
IL-8, whereas homogenates from anti-IL-8-treated animals were reduced
more than 10-fold (0.70 ± 0.01 ng/ml IL-8). Thus, this cannot
account for the inability of this anti-IL-8 antisera to reduce Du145
tumorigenicity. There was no difference in the production of other
measured CXC chemokines between the anti-IL-8- and NGS-treated Du145
cell-injected animals. Given this unexpected result, we hypothesized
that in vivo, Du145 cells use a different CXC chemokine to
regulate tumorigenicity. We reasoned, based on the correlations of CXC
chemokine expression with tumor growth (Figure 4)
, that the most likely
angiogenic CXC chemokine candidate, other than IL-8, to be mediating
Du145 tumorigenicity would be GRO-. As shown in Figure 4
, GRO-
production positively correlated with Du145 tumor growth in
vivo. However, production of ENA-78 and GRO- did not. Based on
these observations and the knowledge that IL-8 was not a contributing
factor to Du145 tumorigenesis, we hypothesized that Du145 cells may use
GRO- to contribute to their tumorigenic phenotype in
vivo.
Du145 Prostate Cancer Cells Tumorigenicity Is Dependent on GRO-
not IL-8
To determine whether GRO- production in vivo by
Du145 cells was contributing to Du145 tumorigenesis, we again used the
strategy of a neutralizing antisera to GRO- to deplete Du145 GRO-
production in vivo. Male SCID mice were injected
subcutaneously with 1 x 106
Du145 cells on
each rear flank. At time of cell innoculum, half of the animals were
given a 0.5-ml injection of a rabbit polyclonal anti-human GRO-
antisera and half were given a 0.5-ml injection of normal rabbit serum
(NRS) as a control. Animals received injections every 48 hours i.p. for
a 5-week period. The Du145 tumor-bearing mice treated with anti-GRO-
antisera displayed a 62% reduction (P = 0.03)
in tumor volume as compared with NRS-treated controls (Figure 7)
. The vascularity index for Du145
tumors treated with NRS was 3503 ± 1570 and for Du145 tumors
treated with anti-GRO-, it was 1876 ± 710, a 47% reduction.
Because in vitro studies had demonstrated that the addition
of recombinant human GRO- or neutralizing antibodies to GRO-
failed to alter the proliferative capacity of Du145 cells in
vitro (Table 1)
, this suggested that the neutralization of
tumor-derived GRO- did not alter the proliferation of the tumor
cells directly but rather reduced the angiogenic potential of the tumor
microenvironment. These data clearly demonstrate that Du145 cells use
GRO-, a distinctly different CXC chemokine than IL-8, to regulate
tumorigenicity in vivo.
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antisera were tested in the CMP assay, three
of five eyes were positive in the Du145 + NRS group. In contrast, tumor
homogenates from the Du145 + anti- GRO- group resulted in a reduced
neovascular response or a negative response (Figure 8)
in four of five corneas tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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has been shown to have both mitogenic and angiogenic
effects on tumor growth.17
Whereas previous reports have
shown a correlation between IL-8 expression and metastatic potential
using in situ hybridization with PC-3
subclones26
or shown IL-8 expression in human prostate
cancer specimens,16
we have now directly demonstrated that
human prostate cancer tumorigenicity is positively regulated by
distinct angiogenic CXC chemokines. It is important to note that these
experiments were carried out in SCID mice, thus we have investigated
the tumorigenic potential of these molecules in the absence of immune
effector cell (T- and B-cell) infiltration. Because these studies used
whole Ig to neutralize the CXC chemokines in vivo, one
formal possibility to explain the reduced tumor growth could be
formation of immune complexes with subsequent ADCC via nonimmune
inflammatory cells within the tumor microenviornment. However, a few
observations make this hypothesis unlikely. The first is that previous
experiments comparing neutralizing or control Ab-treated mice with
untreated mice show no histological or tumor volume differences between
control Ab-treated and untreated mice, suggesting that there was no
obvious immune complex formation. Another observation is that there is
no prominent inflammatory cell infiltration seen in these tumors
histologically, again suggesting there is no accumulation of ADCC
effector cells in this model system. Given that ELR-positive CXC
chemokines are known to be neutrophil chemoattractants, it is
interesting that there is no marked neutrophil influx in these tumors
histologically. This is perhaps explained by the fact that these tumors
are secreting high enough circulating levels of these chemokines that
the neutrophils are desensitized and unable to sense a local
chemotactic gradient. This simulates the phenomenon recently described
in the IL-8 transgenic mouse.27
We have shown that the prostate cancer cell lines, PC-3 and Du145
constitutively express higher levels of angiogenic CXC chemokines than
do LNCaP cells and grow progressively in SCID mice. Furthermore, a
normal prostate epithelial cell line (PrEC) obtained from Clonetics
(San Diego, CA) cultured for 96 hours produced only 0.5 ng/ml IL-8
(25-fold less than PC-3). Futhermore, levels of GRO-, ENA-78, and
GRO- were all reduced by at least 20-fold in PrEC as compared with
PC-3, suggesting that consitutive over-expression of angiogenic CXC
chemokines may be a marker of neoplastic transformation. In addition,
we have demonstrated that these molecules work as angiogenic factors
for tumor neovascularization and not as mitogenic factors for the tumor
cells directly. In contrast, the LNCaP prostate cancer cell line, which
does not express high levels of angiogenic CXC chemokines, does not
form subcutaneous tumors in SCID mice. These observations suggest that
CXC chemokine production contributes to the overall tumorigenicity of
prostate cancer in this model. However, different prostate cancer cell
lines appear to use distinct angiogenic CXC chemokines to modulate
their tumorigenicity. The PC-3 cell line uses IL-8, whereas the Du145
cell line uses GRO-. Interestingly, the Du145 cell line expresses
IL-8 at high levels constitutively in vitro but seems to
express GRO- at higher levels in vivo. Furthermore, the
PC-3 cell line produces GRO- and GRO- both in vitro
and in vivo, however, these molecules fail to demonstrate a
direct correlation with tumor growth in vivo. These data
suggest that it may be difficult to determine by in vitro
measurements alone which angiogenic CXC chemokines may be important
in vivo. Furthermore, CXC chemokine expression alone may not
dictate tumorigenic potential per se. It is likely that other
metastatic factors such as adhesion molecules and matrix
metalloproteinases must also be expressed for successful tumor growth
and metastases.
We have now shown that different cancer cell lines from the same
general cancer type can use distinct angiogenic CXC chemokines. This
difference in utilization may severely limit the usefulness of
antineovascular therapies aimed at neutralizing angiogenic CXC
chemokine ligands (eg, neutralizing IL-8 Abs). Instead, these data
suggest that a better alternative for therapeutic intervention may be
to block the action of multiple angiogenic CXC chemokines by using
either receptor antagonists or agents that directly inhibit
angiogenesis. The receptor for all angiogenic CXC chemokines appears to
be CXCR2. Interestingly, this receptor is present not only on tumor
cells but also tumor-associated endothelial cells in
melanoma.17
Furthermore, this receptor is associated with
head and neck cancers as well as breast cancer .28,29
Immunotherapy aimed at blocking CXCR2 is attractive because it could
potentially limit the angiogenic signal from all angiogenic CXC
chemokines. The only caveat to this is that in humans there are two
receptors for IL-8. The second is CXCR1, a high affinity receptor for
IL-8, which is relatively specific for IL-8, and does not bind most
other ELR-positive angiogenic CXC chemokines.30
The
contribution of this receptor to IL-8-mediated tumorigenicity cannot be
modeled in mice because there is no CXCR1 homologue in
mice.31
However, the angiogenic signaling of the CXCR1
receptor is questionable because our preliminary data has shown that
CXCR2 Ab blocks human endothelial cell chemotaxis to IL-8 in
vitro, whereas CXCR1 Ab does not (unpublished observation). Of
interest, the PC-3 and Du145 cell lines had undetectable levels of
CXCR1 and CXCR2 cell surface staining by FACS analysis, whereas human
neutrophils were strongly positive for both receptors.
Another potential therapy for tumor-induced angiogenesis may be to block the action with angiostatic molecules. In this regard, it is interesting that there are also angiostatic members of the CXC chemokine family. The angiogenic CXC chemokines all share the Glu-Leu-Arg motif (the ELR motif) immediately preceding the first conserved cysteine amino acid in the primary structure. All CXC chemokine members which do not have the ELR motif are, in fact, angiostatic.10 The angiostatic members include the molecules PF4, IP-10, and MIG. Interestingly, the PC-3 and Du145 cell lines show very low levels of these molecules when analyzed either in vitro or in vivo (data not shown). Furthermore, because the antichemokine Ab treatment never diminished tumor growth completely, it is reasonable to assume that other, nonchemokine, angiogenic factors may be present. IL-6 has been documented to be a paracrine growth factor for LNCaP cells and an autorcrine growth factor for both PC-3 and Du145 cells, which express the IL-6 receptor.32,33 Because IL-6 is not an angiogenic factor, it is possible that a combination therapy aimed at eliminating the mitogenic effects of IL-6 as well as the angiogenic effects of the CXC chemokines could be more efficacious. Furthermore, although the LNCaP cell line alone does not form tumors in SCID mice when grown subcutaneously, other investigators have reported significant androgen-dependent LNCaP cell growth in 88% of nude mice when LNCaP cells were injected subcutaneously with 0.25 ml of Matrigel.34 It would be interesting to see which angiogenic factors are predominating in this instance. Two likely candidates may be VEGF and bFGF. In fact, VEGF and bFGF have been demonstrated in both PC-3 and Du145 cells previously,16,26,35 and LNCaP growth in Matrigel has been shown to be augmented by bFGF.36 Interestingly, bFGF, but not VEGF, has been shown to be expressed at higher levels in highly metastatic subclones of prostate cancer cell lines.26 Using in situ hybridization and immunohistochemistry, we have found that IL-8 is present in a higher percentage of NSCLC tumor cells in a metastatic lesion than in the primary lesion18 (unpublished observation). It will be interesting to determine whether this observation holds true for prostate cancer as well. Taken together, these data would support the notion that angiogenic CXC chemokines are important angiogenic factors regulating both primary tumorigenesis as well as metastasis and suggest that therapeutic strategies aimed at antagonizing CXC chemokine action should be beneficial in treating many solid tumors.
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Footnotes |
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Supported by Grants CA79046 (B. B. Moore), CA72543 (D. A. Arenberg), and CA66180, P50HL56402, and P50HL60289 (R. M. Strieter).
Accepted for publication February 18, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med 1996, 184:981-992This article has been cited by other articles:
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  I. Kogan-Sakin, M. Cohen, N. Paland, S. Madar, H. Solomon, A. Molchadsky, R. Brosh, Y. Buganim, N. Goldfinger, H. Klocker, et al. Prostate stromal cells produce CXCL-1, CXCL-2, CXCL-3 and IL-8 in response to epithelia-secreted IL-1 Carcinogenesis, April 1, 2009; 30(4): 698 - 705. [Abstract] [Full Text] [PDF]
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 E. C. Keeley, B. Mehrad, and R. M. Strieter Chemokines as Mediators of Neovascularization Arterioscler. Thromb. Vasc. Biol., November 1, 2008; 28(11): 1928 - 1936. [Abstract] [Full Text] [PDF]
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 H. Kawanishi, Y. Matsui, M. Ito, J. Watanabe, T. Takahashi, K. Nishizawa, H. Nishiyama, T. Kamoto, Y. Mikami, Y. Tanaka, et al. Secreted CXCL1 Is a Potential Mediator and Marker of the Tumor Invasion of Bladder Cancer Clin. Cancer Res., May 1, 2008; 14(9): 2579 - 2587. [Abstract] [Full Text] [PDF]
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 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]
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 Y. Lu, Z. Cai, G. Xiao, E. T. Keller, A. Mizokami, Z. Yao, G. D. Roodman, and J. Zhang Monocyte Chemotactic Protein-1 Mediates Prostate Cancer-Induced Bone Resorption Cancer Res., April 15, 2007; 67(8): 3646 - 3653. [Abstract] [Full Text] [PDF]
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 M. R. McClelland, S. L. Carskadon, L. Zhao, E. S. White, D. G. Beer, M. B. Orringer, A. Pickens, A. C. Chang, and D. A. Arenberg Diversity of the Angiogenic Phenotype in Non-Small Cell Lung Cancer Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 343 - 350. [Abstract] [Full Text] [PDF]
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S. J. Desai, A.-H. Ma, C. G. Tepper, H.-W. Chen, and H.-J. Kung Inappropriate Activation of the Androgen Receptor by Nonsteroids: Involvement of the Src Kinase Pathway and Its Therapeutic Implications Cancer Res., November 1, 2006; 66(21): 10449 - 10459. [Abstract] [Full Text] [PDF]
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Y. Wen, S. F. Giardina, D. Hamming, J. Greenman, E. Zachariah, M. D. Bacolod, H. Liu, J. Shia, P. S. Amenta, F. Barany, et al. GRO{alpha} Is Highly Expressed in Adenocarcinoma of the Colon and Down-Regulates Fibulin-1. Clin. Cancer Res., October 15, 2006; 12(20): 5951 - 5959. [Abstract] [Full Text] [PDF]
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A. Yano, Y. Fujii, A. Iwai, Y. Kageyama, and K. Kihara Glucocorticoids Suppress Tumor Angiogenesis and In vivo Growth of Prostate Cancer Cells. Clin. Cancer Res., May 15, 2006; 12(10): 3003 - 3009. [Abstract] [Full Text] [PDF]
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M. Caunt, L. Hu, T. Tang, P. C. Brooks, S. Ibrahim, and S. Karpatkin Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis. Cancer Res., April 15, 2006; 66(8): 4125 - 4132. [Abstract] [Full Text] [PDF]
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D. S. Michaud, S. E. Daugherty, S. I. Berndt, E. A. Platz, M. Yeager, E. D. Crawford, A. Hsing, W.-Y. Huang, and R. B. Hayes Genetic Polymorphisms of Interleukin-1B (IL-1B), IL-6, IL-8, and IL-10 and Risk of Prostate Cancer. Cancer Res., April 15, 2006; 66(8): 4525 - 4530. [Abstract] [Full Text] [PDF]
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 B. N. Gomperts and R. M. Strieter CXC Chemokines in Angiogenesis and Metastases Am. Assoc. Cancer Res. Educ. Book, April 1, 2006; 2006(1): 11 - 18. [Full Text] [PDF]
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B. Wang, D. T. Hendricks, F. Wamunyokoli, and M. I. Parker A Growth-Related Oncogene/CXC Chemokine Receptor 2 Autocrine Loop Contributes to Cellular Proliferation in Esophageal Cancer. Cancer Res., March 15, 2006; 66(6): 3071 - 3077. [Abstract] [Full Text] [PDF]
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 H. Shen, R. Schuster, K. F. Stringer, S. E. Waltz, and A. B. Lentsch The Duffy antigen/receptor for chemokines (DARC) regulates prostate tumor growth FASEB J, January 1, 2006; 20(1): 59 - 64. [Abstract] [Full Text] [PDF]
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Y. Zhou, J. Zhang, Q. Liu, R. Bell, D. A. Muruve, P. Forsyth, M. Arcellana-Panlilio, S. Robbins, and V.W. Yong The chemokine GRO-{alpha} (CXCL1) confers increased tumorigenicity to glioma cells Carcinogenesis, December 1, 2005; 26(12): 2058 - 2068. [Abstract] [Full Text] [PDF]
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 J. Mestas, M. D. Burdick, K. Reckamp, A. Pantuck, R. A. Figlin, and R. M. Strieter The Role of CXCR2/CXCR2 Ligand Biological Axis in Renal Cell Carcinoma J. Immunol., October 15, 2005; 175(8): 5351 - 5357. [Abstract] [Full Text] [PDF]
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F. Zhang, J. Lee, S. Lu, C. A. Pettaway, and Z. Dong Blockade of Transforming Growth Factor-{beta} Signaling Suppresses Progression of Androgen-Independent Human Prostate Cancer in Nude Mice Clin. Cancer Res., June 15, 2005; 11(12): 4512 - 4520. [Abstract] [Full Text] [PDF]
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 J. Huang, J. L. Yao, L. Zhang, P. A. Bourne, A. M. Quinn, P. A. di Sant'Agnese, and J. E. Reeder Differential Expression of Interleukin-8 and Its Receptors in the Neuroendocrine and Non-Neuroendocrine Compartments of Prostate Cancer Am. J. Pathol., June 1, 2005; 166(6): 1807 - 1815. [Abstract] [Full Text] [PDF]
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C. Murphy, M. McGurk, J. Pettigrew, A. Santinelli, R. Mazzucchelli, P. G. Johnston, R. Montironi, and D. J.J. Waugh Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer Clin. Cancer Res., June 1, 2005; 11(11): 4117 - 4127. [Abstract] [Full Text] [PDF]
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Y.-M. Wu, D. R. Robinson, and H.-J. Kung Signal Pathways in Up-regulation of Chemokines by Tyrosine Kinase MER/NYK in Prostate Cancer Cells Cancer Res., October 15, 2004; 64(20): 7311 - 7320. [Abstract] [Full Text] [PDF]
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E. Lavergne, C. Combadiere, M. Iga, A. Boissonnas, O. Bonduelle, M. Maho, P. Debre, and B. Combadiere Intratumoral CC Chemokine Ligand 5 Overexpression Delays Tumor Growth and Increases Tumor Cell Infiltration J. Immunol., September 15, 2004; 173(6): 3755 - 3762. [Abstract] [Full Text] [PDF]
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S. Halin, P. Wikstrom, S. H. Rudolfsson, P. Stattin, J. A. Doll, S. E. Crawford, and A. Bergh Decreased Pigment Epithelium-Derived Factor Is Associated with Metastatic Phenotype in Human and Rat Prostate Tumors Cancer Res., August 15, 2004; 64(16): 5664 - 5671. [Abstract] [Full Text] [PDF]
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G. G. Vaday, S.-B. Hua, D. M. Peehl, M. H. Pauling, Y.-H. Lin, L. Zhu, D. M. Lawrence, H. D. Foda, and S. Zucker CXCR4 and CXCL12 (SDF-1) in Prostate Cancer: Inhibitory Effects of Human Single Chain Fv Antibodies Clin. Cancer Res., August 15, 2004; 10(16): 5630 - 5639. [Abstract] [Full Text] [PDF]
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E. V. Bobrovnikova-Marjon, P. L. Marjon, O. Barbash, D. L. Vander Jagt, and S. F. Abcouwer Expression of Angiogenic Factors Vascular Endothelial Growth Factor and Interleukin-8/CXCL8 Is Highly Responsive to Ambient Glutamine Availability: Role of Nuclear Factor-{kappa}B and Activating Protein-1 Cancer Res., July 15, 2004; 64(14): 4858 - 4869. [Abstract] [Full Text] [PDF]
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 H. Shen and A. B. Lentsch Progressive dysregulation of transcription factors NF-{kappa}B and STAT1 in prostate cancer cells causes proangiogenic production of CXC chemokines Am J Physiol Cell Physiol, April 1, 2004; 286(4): C840 - C847. [Abstract] [Full Text] [PDF]
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 A. Prasad, A. Z. Fernandis, Y. Rao, and R. K. Ganju Slit Protein-mediated Inhibition of CXCR4-induced Chemotactic and Chemoinvasive Signaling Pathways in Breast Cancer Cells J. Biol. Chem., March 5, 2004; 279(10): 9115 - 9124. [Abstract] [Full Text] [PDF]
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M. P. Keane, J. A. Belperio, Y. Y. Xue, M. D. Burdick, and R. M. Strieter Depletion of CXCR2 Inhibits Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer J. Immunol., March 1, 2004; 172(5): 2853 - 2860. [Abstract] [Full Text] [PDF]
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E. Lavergne, B. Combadiere, O. Bonduelle, M. Iga, J.-L. Gao, M. Maho, A. Boissonnas, P. M. Murphy, P. Debre, and C. Combadiere Fractalkine Mediates Natural Killer-Dependent Antitumor Responses in Vivo Cancer Res., November 1, 2003; 63(21): 7468 - 7474. [Abstract] [Full Text] [PDF]
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F. Zhang, W. Lu, and Z. Dong Tumor-infiltrating Macrophages Are Involved in Suppressing Growth and Metastasis of Human Prostate Cancer Cells by INF-{beta} Gene Therapy in Nude Mice Clin. Cancer Res., September 1, 2002; 8(9): 2942 - 2951. [Abstract] [Full Text] [PDF]
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A. B. LENTSCH The Duffy antigen/receptor for chemokines (DARC) and prostate cancer. A role as clear as black and white? FASEB J, July 1, 2002; 16(9): 1093 - 1095. [Abstract] [Full Text] [PDF]
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S. L. McCarron, S. Edwards, P. R. Evans, R. Gibbs, D. P. Dearnaley, A. Dowe, C. Southgate, D. F. Easton, R. A. Eeles, and W. M. Howell Influence of Cytokine Gene Polymorphisms on the Development of Prostate Cancer Cancer Res., June 1, 2002; 62(12): 3369 - 3372. [Abstract] [Full Text] [PDF]
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T. Karashima, P. Sweeney, J. W. Slaton, S. J. Kim, D. Kedar, J. I. Izawa, Z. Fan, C. Pettaway, D. J. Hicklin, T. Shuin, et al. Inhibition of Angiogenesis by the Antiepidermal Growth Factor Receptor Antibody ImClone C225 in Androgen-independent Prostate Cancer Growing Orthotopically in Nude Mice Clin. Cancer Res., May 1, 2002; 8(5): 1253 - 1264. [Abstract] [Full Text] [PDF]
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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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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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 J. A. Belperio, M. P. Keane, D. A. Arenberg, C. L. Addison, J. E. Ehlert, M. D. Burdick, and R. M. Strieter CXC chemokines in angiogenesis J. Leukoc. Biol., July 1, 2000; 68(1): 1 - 8. [Abstract] [Full Text]
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K. Inoue, J. W. Slaton, B. Y. Eve, S. J. Kim, P. Perrotte, M. D. Balbay, S. Yano, M. Bar-Eli, R. Radinsky, C. A. Pettaway, et al. Interleukin 8 Expression Regulates Tumorigenicity and Metastases in Androgen-independent Prostate Cancer Clin. Cancer Res., May 1, 2000; 6(5): 2104 - 2119. [Abstract] [Full Text]
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K. Inoue, J. W. Slaton, S. J. Kim, P. Perrotte, B. Y. Eve, M. Bar-Eli, R. Radinsky, and C. P. N. Dinney Interleukin 8 Expression Regulates Tumorigenicity and Metastasis in Human Bladder Cancer Cancer Res., April 1, 2000; 60(8): 2290 - 2299. [Abstract] [Full Text]
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