| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics, Preclinical Pharmacology |
Departments of Cancer Biology [K. I., J. W. S., D. W. D., C. J. B., D. M., R. R., C. P. N. D.] and Urology [P. P., P. S., C. P. N. D.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and ImClone Systems, New York, New York 10014 [D. J. H.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
54,000 persons and results in 12,000 deaths annually
(1)
. Clinical studies evaluating the significance of EGFR
expression in human TCC have shown that >50% of human TCCs
overexpress EGFR and that the level of expression directly correlates
with tumor grade, stage, and survival (2, 3, 4, 5, 6)
. In
superficial bladder cancer, EGFR expression correlates with
multiplicity, time to disease recurrence, and overall recurrence rates
(2, 3, 4)
. EGFR expression also predicts for disease
progression from superficial to muscle-invasive or metastatic TCC and
is an independent prognostic factor for death from superficial TCC
(2
, 5
, 6)
. EGFR expression is also an important prognostic
factor for patients with advanced TCC, and patients with
muscle-invasive TCCs that overexpress EGFR have only a 20% probability
of long-term cancer-specific survival (3, 4, 5, 6)
. These
results have shown the importance of EGFR expression for the growth,
invasion, and subsequent metastasis of TCC. We recently reported results of a study in which human bladder TCC established orthotopically in nude mice was treated with the anti-EGFR monoclonal antibody C225 (7) . In this study, C225 inhibited tumor growth and metastasis by inhibiting tumor-induced neovascularization by down-regulating expression of the angiogenic factors VEGF, IL-8, and bFGF by the tumor cells. Treatment with C225 also down-regulated expression of MMP-9 and induced apoptosis within the regressing tumors.
Previous studies have shown that therapy with C225 or the anti-Her2/nev receptor antibody interceptor was enhanced by combination with cisplatin (8 , 9) , topotecan (10) , doxorubicin (11 , 12) , or paclitaxel (12) . Paclitaxel inhibits cell replication by enhancing polymerization of tubulin monomers into stabilized microtubule bundles that are unable to reorganize into the proper structures for mitosis (13 , 14) , resulting in cell cycle blockade in mitosis and subsequent activation of an apoptotic pathway (15 , 16) . Paclitaxel has also been shown to be an active agent for the treatment of advanced TCC (17 , 18) . We therefore evaluated the antitumor effect of C225 and paclitaxel used together as therapy for metastatic TCC growing within the bladder of athymic nude mice.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Reagents.
The chimeric (mouse-human) anti-EGFR monoclonal antibody C225 was
generously provided by ImClone Systems, Inc. (New York, NY). Paclitaxel
was purchased from Bristol-Myers Squibb Co. (Princeton, NJ).
In Vitro Growth Inhibition.
We evaluated the in vitro dose-dependent antiproliferative
effect of C225 and paclitaxel by incubating 1 x
103
253J B-V cells for 24 h in serum-free
medium, and then changing the medium to 10% FBS-supplemented MEM
containing increasing concentrations of C225 (0–1000 µg/ml) and/or
paclitaxel (0–1000 µg/ml), or both. Growth inhibition was determined
after 72 h by [3
H]thymidine incorporation.
In Vitro Apoptosis.
To evaluate the in vitro dose-dependent apoptotic effect of
C225 and paclitaxel, we incubated 5 x 105
253J B-V cells for 24 h in serum-free medium and then in 10%
FBS-supplemented MEM containing increasing concentrations of C225
(0–1000 µg/ml) and/or paclitaxel (0–1000 µg/ml), or both. Cells
were harvested by centrifugation and stained by incubation at
4°C for 24 h in PBS containing 50 µg/ml PI. DNA
fragmentation was quantified by flow cytometry with PI staining.
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
air-flow 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 253J-BV cells (60–70% confluent) were prepared for injection
as described previously (19)
. Mice were anesthetized with
methoxyflurane. A lower midline incision was made, and viable tumor
cells (1 x 106 cells in 0.05 ml of HBSS)
were orthotopically implanted into the bladder wall on day 0. 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.
In Vivo Treatment of Established Human TCC Growing
in the Bladders of Athymic Nude Mice.
Treatment began on day 21. Tumors were harvested from a group of
controls (n = 5) on day 21 prior to initiation of
therapy. Mice were randomly separated into four groups
(n = 10) and treated for 4 weeks according to the
schedule shown in Fig. 1
. Mice then were killed and necropsied on day 56. 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 quickly frozen in liquid nitrogen for mRNA
extraction, fixed in 10% buffered formalin, placed in OCT compound
(Miles Laboratories, Elkhart, IN), and frozen at -80°C or
mechanically dissociated and put into tissue culture. The lungs and
lymph nodes were fixed in 10% buffered formalin or mechanically
dissociated and put into tissue culture.
|
A poly(dT)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 chemistry (Research Genetics, Huntsville, AL). The lyophilized probes were reconstituted to a stock solution at 1 µg/µl in 10 mM Tris (pH 7.6), 1 mM EDTA. Immediately before use, the stock solution was diluted with probe dilution (Research Genetics).
In situ mRNA hybridization was performed as described previously with minor modifications (25 , 26) . ISH was performed using the Microprobe Manual Staining System (Fisher Scientific, Pittsburgh, PA; Ref. 27 ). Tissue sections (4 µm) of formalin-fixed, paraffin-embedded specimens were mounted on silane-treated ProbeOn slides (Fisher Scientific; Refs. 25 , 26 ). 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 to enhance a weak reaction, samples were incubated a second time with fresh chromogen substrate. A red stain indicated a positive reaction. Control for endogenous alkaline phosphatase included treatment of the sample in the absence of the biotinylated probe and use of chromogen alone.
Quantification of Color Reaction.
Stained sections were examined using 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 Optimas image analysis software (version 4.10;
Media Cybernetics, Silver Spring, MD). 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, so 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(dT)20. The results were presented as the
intensity of expression for each treatment group compared with the
control, which was set to 100 (7)
.
IHC.
For IHC 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 containing PBS (pH 7.5), 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 one of the
following: a 1:100 dilution of rat monoclonal anti-CD31 antibody
(PharMingen, San Diego, CA; Ref. 28
), 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/vascular permeability factor antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), a 1:50 dilution of a rabbit polyclonal
anti-IL-8 antibody (Biosource International, Camarillo, CA), a 1:100
dilution of mouse monoclonal anti-MMP-9 antibody (Oncogene Research
Products, Cambridge, MA), or a 1:100 dilution of mouse monoclonal
anti-PCNA antibody (DAKO, Carpinteria, CA). 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, West Grove, PA), antirabbit IgG,
F(ab)2 fragment (Jackson ImmunoResearch
Laboratory), antimouse IgG1 (PharMingen), or antimouse IgG (Jackson
ImmunoResearch Laboratory). 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 using a water and
alcohol-based mounting medium (Universal mount, Research Genetics).
Quantification of Intensity of Immunostaining.
The intensity of the immunostaining of bFGF, VEGF, IL-8, and MMP-9 was
quantified in three different areas of each sample by an image analyzer
using Optimas image analysis software program (Media Cybernetics) to
obtain an average measurement. Results are presented as a ratio between
the expression by the tumor and by normal mucosa (which was set at 100;
Ref. 7
).
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. (29)
. Clusters of stained endothelial
cells distinct from adjacent microvessels, tumor cells, or other
stromal cells were counted as one microvessel. The tissue was
photographed using a cooled CCD Optotronics Tec 470 camera (Optotronics
Engineering, Goletha, CA) linked to a computer and digital printer
(Sony Corporation). The MVD was expressed as the average number of the
five highest areas identified within a single x200 field.
TUNEL Assay.
For the TUNEL assay, 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 distilled water with Brij and treated with a 1:500 dilution
of proteinase K (20 µg/ml) for 15 min; endogenous peroxidase was
blocked using 3% hydrogen peroxide in PBS for 12 min. The samples were
washed three times with distilled water with Brij and incubated for 10
min at room temperature with TdT buffer. Excess TdT buffer was drained,
and the samples were incubated for 18 h at 4°C with terminal
transferase and biotin-16-dUTP. The samples were then rinsed four times
with Tris-buffer and incubated for 30 min at 37°C with a 1:400
dilution of peroxidase-conjugated streptavidin. 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), and
washed three times with PBS. The slides were mounted using a Universal
Mount (Research Genetics).
Quantification of Cell Proliferation and Apoptosis.
Cell proliferation and apoptosis were determined by IHC of tissue
sections with anti-PCNA antibodies and TUNEL assay. The tissue was
photographed using a cooled CCD Optotronics Tec 470 camera
linked to a computer and digital printer. The density of
proliferative cells and apoptotic cells was expressed as an average
number of the five highest areas identified within a single x200
field.
Immunofluorescence Double-Staining of Endothelial Cells and
Apoptotic Cells.
Frozen tissue sections (8 mm thick) were fixed with cold acetone for 5
min, acetone and chloroform (1:1, v/v) for 5 min, and acetone
for 5 min. Samples were washed three times with PBS and incubated with
protein blocking solution containing 5% normal horse serum and 1%
normal goat serum in PBS for 20 min. The blocking solution was drained,
and the samples were incubated with a 1:400 dilution of rat monoclonal
anti-CD31 antibody (PharMingen; Ref. 29
) for 24 h at
4°C. The samples were then rinsed with PBS three times for 3 min and
incubated with protein-blocking solution for 10 min at room
temperature. The blocking solution was again drained, and the samples
were incubated for 1 h at room temperature with a 1:200 dilution
of secondary goat antirat conjugated to Texas Red (Jackson
ImmunoResearch Laboratory). The samples were then washed twice with PBS
containing 0.1% Brij and then washed with PBS for 5 min. The TUNEL
assay was performed using a commercial kit (Promega, Madison, WI) with
the following modifications. The samples were fixed with 4%
paraformaldehyde for 10 min at room temperature. The samples were then
washed with PBS twice for 5 min and incubated with 0.2% Triton X-100
for 15 min at room temperature. The samples were again washed with PBS
twice (5 min each time) and incubated with equilibration buffer (from
the kit) for 10 min at room temperature. The equilibration buffer was
drained, and reaction buffer containing equilibration buffer,
nucleotide mixture, and TdT enzyme was added to the tissue sections and
incubated in a humid atmosphere at 37°C for 1 h, avoiding
exposure to light. The reaction was terminated by immersing the samples
in 2x SSC for 15 min. The samples were then washed three times (5 min
each time) to remove unincorporated fluorescein-dUTP. For
quantification of endothelial cells, the samples were incubated with
300 mg/µl Hoechst stain for 10 min at room temperature. The
samples were then washed with PBS twice (5 min each time). To preserve
fluorescence and reduce bleaching, Prolong solution (Molecular Probes,
Inc., Eugene, OR) was used to mount coverslips. The slides were
examined using an inverted microscope (model IX70; Olympus, Melville,
NY). Endothelial cells were identified by red fluorescence, and DNA
fragmentation was detected by localized green fluorescence within the
nuclei of apoptotic cells. Results were expressed as the ratio of
apoptotic endothelial cells to total endothelial cells in 10 random
fields at x40 magnification.
Statistical Analysis.
The statistical differences for the number of vessels, proliferative
cells, and apoptotic cells and for staining intensity for EGFR and
activated EGFR, bFGF, VEGF, IL-8, and MMP-9 within the bladder tumors
were analyzed by the Mann-Whitney test. Incidences of tumor and
metastasis were statistically analyzed by the
2 test. P < 0.05 was
considered significant.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A). At low concentrations, C225 (1.0 and 10 µg/ml) enhanced
the antiproliferative effects of paclitaxel (Fig. 2
B).
|
.
|
. Treatment with paclitaxel followed 2 days later by C225 (median
bladder weight, 85 mg; range, 69–133 mg), was significantly
more effective at eradicating bladder tumors than was treatment with
C225 alone (median bladder weight, 168 mg; range, 72–288 mg;
P < 0.05), paclitaxel alone (median bladder weight,
273 mg; range, 83–563 mg; P < 0.005) or PBS alone
(median bladder weight, 481 mg; range, 377–877 mg; P < 0.0005). Combination therapy also significantly reduced spontaneous
lymph node metastasis (no animal had metastases) compared with
treatment using paclitaxel alone (seven of nine animals;
P < 0.005) or PBS alone (nine of nine animals;
P < 0.0001). Although two of seven mice treated with
C225 alone developed spontaneous lymph node metastases, this result did
not significantly differ from results following combination therapy.
|
and Fig. 4
) and IHC (Table 2
and Fig. 5
), respectively. The mRNA and protein expression of bFGF, VEGF, IL-8,
and MMP-9 was 40–70% lower in tumors of mice treated with C225 alone
or with C225 in combination with paclitaxel, compared with PBS-treated
controls (P < 0.005; Table 2
and Fig. 4
).
|
|
|
.
Enhancement of Apoptosis and Inhibition of Proliferation by Therapy
with C225 and Paclitaxel.
We evaluated the effect of therapy with C225 and paclitaxel on cellular
proliferation and apoptosis by IHC for PCNA and TUNEL, respectively.
The number of PCNA-positive cancer cells per x200 field was
significantly lower in tumors from mice treated with paclitaxel alone
(70 ± 11) or with C225 alone (87 ± 16) than in tumors from
PBS-treated controls (248 ± 68; P < 0.005). The
combination of C225 and paclitaxel significantly inhibited
proliferation compared with the use of each agent alone, with the
greatest difference seen after initial therapy with paclitaxel,
followed by C225 (30 ± 9; P < 0.005).
The number of apoptotic cancer cells per x200 field significantly
increased, from 4 ± 1 in PBS-treated controls to 14 ± 4 and 11 ± 2 following therapy with C225 and paclitaxel,
respectively (P < 0.005; Table 3
and Fig. 6
).
|
|
. Endothelial cells were detected by localized red
fluorescence, whereas green fluorescence was detected within the nuclei
of apoptotic cells. Double-labeling of endothelial cells undergoing
apoptosis resulted in localized yellow fluorescence. The number of
apoptotic endothelial cells counted per x400 field was calculated as
the ratio of double-labeled to total endothelial cells. We observed
significantly higher apoptotic indices for endothelial cells after
treatment with either paclitaxel or C225 (P < 0.005).
Treatment with paclitaxel and C225 resulted in an increase in the
apoptotic index to 49.5 ± 11.3, which was significantly higher
than values for all other therapy groups (P < 0.005;
Table 4
). Clusters of apoptotic tumor cells were seen surrounding apoptotic
endothelial cells when the two agents were combined (Fig. 6)
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Recent reports indicate that biological agents such as antagonists of EGFR or HER-2/neu receptor modulate host responses and enhance the efficacy of standard cytoreductive chemotherapy (7 , 33) . The anti-EGFR antibody C225 has significant antitumor activity against a number of human malignancies, including those of the prostate (34) , breast (8) , colon (8) , ovary (8) , and kidney (35) , and TCC of the bladder (7) . This antitumor effect is modulated by cell cycle regulation via induction of apoptosis and by inhibition of invasion and tumor-induced angiogenesis (36, 37, 38, 39) . Both C225 and the anti-HER2 monoclonal antibody 4D5 have been combined with doxorubicin and paclitaxel to effectively treat breast and ovarian cancers (8 , 9) . Treatment of human breast xenografts with the HER-2/neu tyrosine kinase inhibitor Emodin sensitized these tumors to paclitaxel and improved the survival of mice treated by this combination. C225 has also been combined with the monoclonal antibody 4D5 to treat ovarian cancer (33) .
In the present study, we evaluated the efficacy of using systemic C225 and paclitaxel to treat human TCC growing within the bladder of nude mice. Paclitaxel demonstrates a significant antitumor effect against TCC of the bladder, both as a single agent and in combination with other cytotoxic agents. Paclitaxel increases microtubule stability by preventing tubulin depolymerization, which results in tubulin bundling (13 , 14) . These cytoskeleton changes lead to cell cycle arrest and apoptosis within 20 h of paclitaxel exposure (40 , 41) . The extent of paclitaxel-induced apoptosis is regulated by Raf-1 activity (42 , 43) , such that 2–4-fold increases in paclitaxel-induced cytotoxicity occur if Raf-1 kinase levels are reduced. Therefore, we treated human TCC growing within the bladders of athymic nude mice with systemic paclitaxel in combination with C225, an agent that down-regulates Raf-1 activity.
In vitro, C225 enhanced the antiproliferative and apoptotic effects of paclitaxel in a dose-dependent manner. The antiproliferative effect of C225 was 25% at a dose of 1 µg/ml, similar to that of paclitaxel at a dose of 0.1 µg/ml When the drugs were combined at that dose, there was an additive effect on cytostasis that approached 55%. Similarly, we observed an additive effect on the induction of apoptosis. Combined use of C225 and paclitaxel at their IC50 doses (100 µg/ml for C225 and 10 µg/ml for paclitaxel) resulted in apoptosis in 64.5% of the tumor cells compared with 50.3% for paclitaxel alone at 10 µg/ml and 8.5% for C225 alone at 100 µg/ml.
The benefit of combining C225 with paclitaxel became more apparent after in vivo treatment. In a previous study (7) , we showed that interruption of EGFR signaling pathways with C225 caused tumor regression and inhibited metastasis of established human TCC growing within the bladders of athymic nude mice. This significant antitumor effect was mediated by the induction of apoptosis and the inhibition of angiogenesis. In the present study, we observed that the cytotoxic and proapoptotic effects of C225 were enhanced by the administration of paclitaxel. This therapy was also very effective in preventing metastasis. Although we could not demonstrate a statistically significant difference compared with treatment using C225 alone, no mice that received initial treatments with paclitaxel followed by C225 developed spontaneous metastasis, whereas two mice treated with C225 alone developed lymph node metastases.
The mechanisms responsible for the interaction of C225 and paclitaxel are unclear. Tumor-induced neovascularization and invasion were inhibited by treatment with C225, secondary to down-regulation of VEGF, bFGF, IL-8, and MMP-9 expression by the 253J B-V tumor cells. Although the down-regulation of invasion and metastasis is an essential function of this therapy, it was not enhanced by the addition of paclitaxel.
It has been hypothesized that therapy with paclitaxel might functionally up-regulate EGFR or the HER2/neu receptor and render cells more susceptible to C225 (44 , 45) . However, although our data indicated that neither EGFR nor activated EGFR is up-regulated by the use of paclitaxel alone, activated EGFR was down-regulated when paclitaxel was administered prior to C225. It is possible that we failed to recognize the functional up-regulation of EGFR because of the time at which we made our measurements (data not shown).
The most likely mechanism responsible for the increased antitumor activity of the combined treatment with C225 and paclitaxel is an increase in apoptosis coupled with a decrease in proliferation. This is the result of the sum of effects on separate pathways regulating apoptosis (15 , 16 , 40 , 41 , 46) . Apoptosis was markedly increased when paclitaxel was administered before C225. We hypothesize that C225-mediated cell cycle arrest limits paclitaxel-induced apoptosis. The ratio of apoptosis to PCNA appears to be a marker that predicts response to this therapy. The ratios in the tumors treated initially with paclitaxel were 4.2-, 6.6-, and 52.1-fold higher than the ratios for paclitaxel or C225 alone and PBS-treated controls, respectively, and 2.4-fold higher than the ratios for initial treatment with C225. Using double-labeling fluorescence, we found that therapy with C225 and paclitaxel induced apoptosis to a lesser degree in endothelial cells as well as tumor cells. This effect became more significant in the group of mice treated initially with paclitaxel, in which we observed clusters of apoptotic tumor cells surrounding apoptotic endothelial cells.
In summary, our experiments demonstrated that paclitaxel enhances the antitumor effect of C225 on human bladder TCC growing within the bladders of athymic nude mice and demonstrated the benefit of combining two therapeutic modalities that have entirely different mechanisms of action. The improved response to this strategy seems to result from enhancement in the induction of apoptosis by paclitaxel and C225.
|
FOOTNOTES |
|---|
1 This work was supported by Core Grant 16672, NIH
Grants CA 67952 (to R. R.) and CA 67914 (to C. P. N. D.), and a
grant from ImClone. 
2 To whom requests for reprints should be
addressed, at Department of Urology, Box 110, The University of Texas
M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX
77030. Phone: (713) 792-3250; Fax: (713) 792-8747. E-mail: cdinney{at}mdanderson.org
3 The abbreviations used are: TCC, transitional
cell carcinoma; EGFR, epidermal growth factor receptor; VEGF, vascular
endothelial cell growth factor; IL-8, interleukin-8; bFGF, basic
fibroblast growth factor; MMP-9, matrix metalloproteinase type 9; FBS,
fetal bovine serum; PI, propidium iodide; ISH, in situ
hybridization; IHC, immunohistochemical staining; PCNA, proliferating
cell nuclear antigen; MVD, microvessel density; TUNEL, terminal
deoxynucleotidyl transferase (TdT)-mediated nick end labeling.
Received 3/14/00; revised 7/13/00; accepted 7/20/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, and epidermal growth factor receptor on the prognosis of superficial bladder cancer. Urology, 51: 645-649, 1998.[CrossRef][Medline]
in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature (Lond.), 394: 485-490, 1998.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  S. L. Emanuel, T. V. Hughes, M. Adams, C. A. Rugg, A. Fuentes-Pesquera, P. J. Connolly, N. Pandey, S. Moreno-Mazza, J. Butler, V. Borowski, et al. Cellular and in Vivo Activity of JNJ-28871063, A Nonquinazoline Pan-ErbB Kinase Inhibitor That Crosses the Blood-Brain Barrier and Displays Efficacy against Intracranial Tumors Mol. Pharmacol., February 1, 2008; 73(2): 338 - 348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Anai, S. Goodison, K. Shiverick, Y. Hirao, B. D. Brown, and C. J. Rosser Knock-down of Bcl-2 by antisense oligodeoxynucleotides induces radiosensitization and inhibition of angiogenesis in human PC-3 prostate tumor xenografts Mol. Cancer Ther., January 1, 2007; 6(1): 101 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shrader, M. S. Pino, G. Brown, P. Black, L. Adam, M. Bar-Eli, C. P.N. Dinney, and D. J. McConkey Molecular correlates of gefitinib responsiveness in human bladder cancer cells Mol. Cancer Ther., January 1, 2007; 6(1): 277 - 285. [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]
|
|
|
|
|
|
H.-J. Lenz, E. Van Cutsem, S. Khambata-Ford, R. J. Mayer, P. Gold, P. Stella, B. Mirtsching, A. L. Cohn, A. W. Pippas, N. Azarnia, et al. Multicenter Phase II and Translational Study of Cetuximab in Metastatic Colorectal Carcinoma Refractory to Irinotecan, Oxaliplatin, and Fluoropyrimidines J. Clin. Oncol., October 20, 2006; 24(30): 4914 - 4921. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Vincenzi, D. Santini, A. Russo, M. Silletta, M. Gavasci, F. Battistoni, G. Di Cuonzo, L. Rocci, N. Gebbia, and G. Tonini Angiogenesis modifications related with cetuximab plus irinotecan as anticancer treatment in advanced colorectal cancer patients Ann. Onc., May 1, 2006; 17(5): 835 - 841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Ettinger Clinical Implications of EGFR Expression in the Development and Progression of Solid Tumors: Focus on Non-Small Cell Lung Cancer. Oncologist, April 1, 2006; 11(4): 358 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Wild, K. Fager, C. Flefleh, D. Kan, I. Inigo, S. Castaneda, F. Luo, A. Camuso, K. McGlinchey, and W. C. Rose Cetuximab preclinical antitumor activity (monotherapy and combination based) is not predicted by relative total or activated epidermal growth factor receptor tumor expression levels Mol. Cancer Ther., January 1, 2006; 5(1): 104 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Inoue, T. Karashima, S. Fukata, A. Nomura, C. Kawada, A. Kurabayashi, M. Furihata, Y. Ohtsuki, and T. Shuin Effect of Combination Therapy with a Novel Bisphosphonate, Minodronate (YM529), and Docetaxel on a Model of Bone Metastasis by Human Transitional Cell Carcinoma Clin. Cancer Res., September 15, 2005; 11(18): 6669 - 6677. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. R. Luo, Z. Yang, H. Dong, A. Camuso, K. McGlinchey, K. Fager, C. Flefleh, D. Kan, I. Inigo, S. Castaneda, et al. Prediction of Active Drug Plasma Concentrations Achieved in Cancer Patients by Pharmacodynamic Biomarkers Identified from the Geo Human Colon Carcinoma Xenograft Model Clin. Cancer Res., August 1, 2005; 11(15): 5558 - 5565. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. N. Younes, O. G. Yigitbasi, Y. W. Park, S.-J. Kim, S. A. Jasser, V. S. Hawthorne, Y. D. Yazici, M. Mandal, B. N. Bekele, C. D. Bucana, et al. Antivascular Therapy of Human Follicular Thyroid Cancer Experimental Bone Metastasis by Blockade of Epidermal Growth Factor Receptor and Vascular Growth Factor Receptor Phosphorylation Cancer Res., June 1, 2005; 65(11): 4716 - 4727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T.C. Chan, M.-M. Hsu, B. C. Goh, E. P. Hui, T.-W. Liu, M. J. Millward, R.-L. Hong, J. Whang-Peng, B. B.Y. Ma, K. F. To, et al. Multicenter, Phase II Study of Cetuximab in Combination With Carboplatin in Patients With Recurrent or Metastatic Nasopharyngeal Carcinoma J. Clin. Oncol., May 20, 2005; 23(15): 3568 - 3576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Raspollini, F. Castiglione, F. Garbini, A. Villanucci, G. Amunni, G. Baroni, V. Boddi, and G. L. Taddei Correlation of Epidermal Growth Factor Receptor Expression with Tumor Microdensity Vessels and with Vascular Endothelial Growth Factor Expression in Ovarian Carcinoma International Journal of Surgical Pathology, April 1, 2005; 13(2): 135 - 142. [Abstract] [PDF]
|
|
|
, ip PBS; 
, ip IM-C225; 
, ip
paclitaxel.
