[CANCER RESEARCH 52, 3776-3781. July 1, 1992]
Advances in Brief
Taxol Blocks Processes Essential for Prostate Tumor Cell (PC-3 ML)
Invasion and Métastases1
Mark E. Stearns2 and Min Wang
Department of Pathology, Medical College of Pennsylvania, Philadelphia, Pennsylvania I9I29
Abstract
We have examined the antimetastatic effects of taxol on a PC-3 human
prostatic tumor variant (PC-3 ML) which metastasizes to the lumbar
vertebrae in severe combined immunodeficiency-carrying (SCID) mice.
Immunofluorescence labeling indicated that taxol (0.5 to 1.0 JUMfor 6 h)
produced an abnormal bundling of microtubules in a dosage-dependent
manner. Slot blotting and gelatinase assays revealed that taxol inhibited
secretion of the M, 72,000 and M, 92,000 type IV collagenases plus a
V/, 57,000 gelatinase. Radioimmunoprecipitation measurements con
firmed that the drug inhibited both the secretion and the synthesis of the
M, 72,000 collagenase. Taxol also blocked total protein secretion but did
not influence total protein synthesis or turnover. Boyden chamber chemotactic studies further showed that taxol (0.5 to 1.0 JIM)inhibited invasion
of Matrigel. More importantly, studies in SCID mice demonstrated that
taxol (50 to 250 mg/mVday) blocked the establishment, growth, and
long-term survival of PC-3 ML cells.
Introduction
Taxol, an alkaloid from Japanese and Pacific yew, is a potent
drug with tremendous potential in the therapeutic treatment of
cancer. Horwitz et al. (1) and others (2) reported that the
principle effect of taxol was to stabilize microtubule structures.
One striking effect of taxol was that it induced an abnormal
bundling of microtubules in cells, blocking cell division (2).
Basically, taxol binds tubulin to form abnormally stable taxol
microtubules (3-5).
In the past few years, taxol has been successfully tested on
patients with cancer (6-12), specifically in the treatment of
malignant melanoma (7) and ovarian carcinoma (8, 9). The
ovarian cancer studies indicated that taxol was active at dosages
of 110 to 175 mg/m3 (8). The drug had significant activity
against melanoma with some non-life-threatening side effects
(i.e., nausea, vomiting) (7). Holmes et al. (2) have recently
shown that the drug can be used to treat metastatic breast
cancer in phase I and II trials. Taxol, therefore, may be of value
in the treatment of a wide range of cancers, including colon and
prostate cancer. Future trials need to assess drug treatment
protocols, optimum dosages, and dose-limiting toxic effects. In
addition, the utility of taxol in treating multidrug-resistant,
hormone-resistant
and metastatic tumor lesions should be
addressed.
In attempts to study prostate cancer métastases,we have
developed a SCID2 mouse model for investigating métastases
to the bone. A variant (PC-3 ML) of human prostate PC-3
Received 1/15/92; accepted 5/11/92.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by NIH-NCI Grant CA53518 to M. E. S.
2To whom requests for reprints should be addressed, at Department of Pa
thology, Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, PA
19129.
3The abbreviations used are: SCID, severe combined immunodeficiency; CM,
conditioned medium.
tumor cells has been isolated which preferentially metastasizes
to the lumbar vertebrae with >80% efficiency following i.v.
injection via the tail vein in SCID mice (13). In this paper, we
have utilized the SCID mouse model to test whether taxol
might have therapeutic utility in the prevention or treatment of
metastatic bone cancer.
We describe novel observations on the antitumorigenic effects
of taxol on biological processes (i.e., protease secretion) impor
tant for tumor cell invasion in vitro. More importantly, we show
that taxol is effective with marginal toxicity in the treatment of
multidrug-resistant, metastatic bone tumors formed by the PC3 ML variant in SCIDs.
Materials
and Methods
The isolation and culturing of human PC-3 cells and DU 145 cells
have been detailed in previous studies (13, 14). The cells were plated
on Matrigel (2 mg/ml) unless indicated differently. We have described
the methods for preparation and use of PC-3 ML CM (15), the Boyden
chamber chemotactic assays, and the preparation and use of Matrigel
(14, 15). The slot blotting and immunofluorescence techniques for
labeling cells also have been developed previously (14, 15). As described
in the table legends, we labeled the cells with [3H]methionine using
standard procedures (13).
For the mouse studies, the cells were injected i.v. and s.c. in SCID
mice using published methods (13). The resulting tumor tissue was
examined by light microscopy following histológica! preparation of
tissue (13). Cryosections were prepared for antibody labeling and im
munofluorescence studies according to the methods described by
Brandtzaeq (16). The monoclonal /î-tubulin
antibodies and polyclonal
M, 72,000 type IV collagenase antibodies used in these studies have
been characterized previously (14, 15). Western blots and slot blots
with the type IV collagenase antibodies used at a dilution of 1:200 (~\
Mg/ml) showed that the antibodies detected as little as 0.10 ng/m\
protease and showed a linear increase in labeling up to about 10 Mg/m'
of purified protease. The levels measured in this paper were within this
linear range.
Gelatinase zymography assays were carried out according to the
procedures of Heussen and Dowdle (17). All protein measurements
were with a Bio-Rad kit using their procedures. All the reagents were
from Sigma Chemical Co. unless stated otherwise.
Taxol (Sigma Chemical Co., St. Louis, MO) was solubili/ed in castor
oil and aliquots (i.e., 5-10 ¿il)
were added to solutions. Similar amounts
of castor oil were added in control experiments. In general, care was
taken to ensure animal welfare on a daily basis.
Toxicity end points were determined by exposing mice to taxol at
increased dosages (25, 50, 100, 200, 250, 300, 350 mg/m2/day) for 5
days. Three mice were tested per dosage. Lethargy, sickness, and
hyperventilation were observed at dosages greater than 250 mg/m2/day
and the animals sacrificed by cervical dislocation. No histopathological
criterion was used to assess toxicity.
Results and Discussion
Micromolar levels of taxol had a dramatic effect on the
microtubule networks of PC-3 ML cells cultured on Matrigel.
3776
ANTIMETASTAT1C
EFFECTS OF TAXOL ON PROSTATE TUMOR
Following immunofluorescence labeling with /3-tubulin antibod
ies, Fig. 1 compares the normal microtubule arrays emanating
from a central microtubule-organizing center in untreated cells
(Fig. IA); and the microtubule bundles observed in the taxoltreated cells (Fig. 15), where the cells were exposed to 0.5 UM
taxol for 6 h at 37°C.Note the discontinuous nature of the
microtubules located in the peripheral margins of the taxolirouted cells.
The dose-dependent effects of taxol on microtubule bundling
were summarized in Table 1. The amount of bundling observed
by immunofluorescence labeling was minimal or nonexistent at
low levels of taxol tested (<0.05 n\t for 1 to 6 h). In contrast,
at higher dosages (>0.5 /¿M
for periods of 6 h) 100% of the cells
exhibited microtubule bundling. A similar response to taxol
was observed in the other PC-3 variants (i.e., 3 x noninvasive)
and DU 145 cells (data not shown). Moreover, these results
Fig. 1. Immunofluorescence images of PC-3 ML cells labeled with /3-tubulin
antibodies and secondar)' antibody conjugated to fluorescein isothiocyanate. A.
untreated; B, 0.5 I/M taxol for 6 h showing microtubule bundles (arrows) in the
cell periphery. A, xlOOO; B, xlSOO. Aliquots (5 M') of castor oil were added to
the untreated cells.
Table 1 Immunofluorescence microscopy studies of microtubule bundling in
taxol-treated PC-3 ML cells
Values show the number of cells (of 100) exhibiting abnormal microtubule
assays in each experiment. Immunolabeling with antibodies for the pi 70 multidrug resistance glycoprotein (antibodies courtesy of Dr. Vic Ling, University of
Toronto) indicated the PC-3 ML variants uniformly expressed this phenotype.
Treatment time
Taxol
(fM)0.01
0.05
0.10
0.50
1.001
h0/100
0/100
5/50
1/100
20/100
33/50
45/100
30/100
50/50
100/100
25/1003h0/5050/506h0/100
100/100
M
Fig. 2. Slot blots with the M, 72,000 type IV collagenase antibodies (1:200
dilution) showing the whole cell extracts (£)and the medium (M) from the same
cells. The cells were (Lane 1) untreated and exposed to taxol for 6 h at (Lane 2)
0.5 fiM and (Lane 3) 1.0 fiM levels. The PC-3 ML cells (passage 5) were seeded
at 2 x 10' cells/ml in 60-mm dishes overnight on Matrigel (2 mg/ml), washed 3
times with Dulbecco's modified Eagle's medium, exposed to drug in Dulbecco's
modified Eagle's medium for 6 h, washed 4 times with Dulbecco's modified
Eagle's medium, and exposed to fresh PC-3 ML CM (10 mg/ml) for 6 h at 37°C
in a 5% CO2 incubator. The medium (3 ml) and whole cell extract (3 ml) from
each dish was collected, concentrated to 0.3 ml by dialysis against PEG 20,000
for 3 to 4 h at 4'C, and blotted (0.3 ml/well) according to published procedures
(15).
23456
Fig. 3. Gelatinase assays (7% gel) demonstrating the relative collagenase levels
(72, =A/r 72,000; 57, M, 57,000) in the medium of PC-3 ML cells untreated
(Lanes 1, 2) or exposed to increased amounts of taxol at (Lanes 3, 4) 0.1 p M or
(Lanes S, 6) 0.5 JIMfor 6 h. The lanes were each loaded with (Lanes 1, 3, 5) 10
fig and (Lanes 2, 4, 6) 20 fig protein, and gels were electrophoresed overnight
(=18 h) at 40 amp/gel. The cells were plated at 2 x IO7 for 3 h. exposed to taxol
for 6 h, washed with serum-free medium, and incubated in serum-free medium
containing 10 mg/ml PC-3 ML CM for 6 h. This medium was harvested and
concentrated 10-fold by dialysis against dry polyethelene glycol powder (M,
20,000) for the zymogram assays.
agree closely with that originally reported for other cells (1, 2).
Therefore, we believe that the limiting dosage-dependent
parameter in these studies was the time for diffusion of taxol
across the cell membranes. Although radiolabeled drug uptake
studies were not done, drug uptake in the cells appeared to be
relatively slow and largely irreversible. For example, microtu
bule bundling was observed following exposure to 0.5 ¿tM
taxol
for 6 h and removal of the drug from the medium for 48 h.
Effects of Taxol on Protease Secretion. Previously, we and
others (13, 15, 17, 18) have reported that type IV collagenase
was secreted by human prostatic tumor cells. Plating these cells
on type IV collagen (14), in the presence of their own CM (13),
significantly elevated the levels of type IV collagenase secreted.
In this paper, the cells were plated on Matrigel (2 mg/ml)
which stimulated the cells to secrete collagenase (i.e., it had a
similar effect as type IV collagen). The inhibitory influence of
taxol on type IV collagenase secretion was then examined by
slot blotting of the culture medium (Fig. 2). Slot blots with
antibodies raised against a A/r 72,000 type IV collagenase
indicated that the protease was normally secreted by PC-3 ML
cells in response to the PC-3 ML CM (10 mg/ml for 6 h). If
the cells were pretreated with taxol (0.5 and 1.0 MMfor 6 h)
prior to stimulation of secretion with conditioned medium, the
drug blocked the secretion of the M, 72,000 type IV collagenase.
Slot blots of whole cell extracts from the same cells confirmed
that type IV collagenase was in fact present in the taxol-treated
PC-3 ML cells (Fig. 2). A standard curve assay with affinitypurified collagenase showed a linear antibody (1:200 dilution)
detection range from 0.01 to 30 ng, with saturation at 30 fig.
Control experiments showed that when l ¿IM
taxol was added
to PC-3 ML CM (10 mg/ml) prior to (for 3 h) and during the
experiment, the drug did not block the ability of CMs to
stimulate protease release (data not shown). We interpret this
to mean that taxol probably does not bind or interfere with
processes activated by factors in the CM perse (14).
Gelatinase assays confirmed that taxol (0.5 pM for 6 h)
inhibited the secretion of two high molecular weight gelatinases,
including a faint band at A/r 92,000 and a pronounced band at
M, 72,000 (Fig. 3). Secretion of lower molecular weight gelati-
3777
ANTIMETASTATIC
EFFECTS OF TAXOL ON PROSTATE TUMOR
Table 2 Pulse labeling experiments measuring effects oflaxol on synthesis
(cpm x 1000 ±SD)
The PC-3 ML cells were seeded at 2 x 10" cells/ml in 10 ml and were exposed
to 0.5 /AI taxol for 0, 6, and 9 h, then pulse labeled with (rani-labeled | 'I l|
methionine (200 ¿iCi/ml;NEN) for 6 h in methionine-free medium, washed 3
limes with SFM," and exposed to SFM containing 10 mg/ml PC-3 ML CM for
6_h.
Taxol treatment
Protein synthesis
Collagenase synthesis
30
Collagenase turnover
28
Collagenase turnover" •¿* 25
Collagenase secretion" 'Oh350
0.1
±5.1
±4.0
±1.5
±0.3
0.6 ±0.1 0.4 ±0.05
±0.2
25 ±0.4
24 ±0.3
±0.4
±0.3
16 ±0.5
17 ±0.3
±0.13h15
12 ±0.46h330
13 ±0.29h295
11 ±0.5
" SFM, serum-free medium.
h The cells were pulse labeled as in the above studies and exposed to SFM
containing 1.0 JIM taxol plus 10 mg/ml PC-3 ML CM for 0, 3, 6, and 9 h.
['H|collagenase was immunoprecipitated from whole cell extracts' and the med
ium'' of the same cells.
Table 3 Effects of taxol on total protein and collagenase secretion
(cpm x 7000 ±SD)
The cells (2 x 106/ml in 10 ml) were labeled (200 ^Ci/ml |'H]methionine for
6 h in methionine-free medium), washed 6 times, exposed to 0.5 MMtaxol for 6
and 9 h. and transferred to serum-free medium containing 10 mg/ml PC-3 ML
conditioned medium for 6 h. Scintillation counting of the medium from each 60mm dish was in triplicate ±SD. Trypan blue (0.1 %) dye exclusion assays indicated
that there was little or no cell death in the presence of taxol.
Exposure
time(h)0
6
9Total
protein
secreted360
±0.20
0.3 ±0.20
0.2 ±0.09Total
showed that the amount of 'H-type IV collagenase [i.e., M,
72,000] immunoprecipitated
from the whole cell extracts of
taxol-treated cells was drastically reduced from that found in
untreated cells [=30,000 ±300 (SD) cpm]. By comparison, the
immunoprecipitates from treated cells contained 600 ±100
cpm after 6 h and 400 ±50 cpm after 9 h exposure. The amount
of total labeled protein in whole cell extracts was 350,000 ±
5,100 cpm in controls, 330,000 ±4,000 cpm after 6 h exposure,
and 295,000 ±1,500 cpm after 9 h exposure to taxol.
To test further whether taxol influenced type IV collagenase
turnover rates, the cells were first labeled (200 nCi/m\ ['H]
methionine for 6 h) and then exposed to 1.0 n\i taxol for 0, 6,
and 9 h. Immunoprecipitation
measurements revealed cytoplasmic counts (cpm) of 28,000 ±220 (at 0 h), 25,000 ±420
(after 6 h), and 24,000 ±330 (after 9 h) incubation in the
presence of taxol. The counts were somewhat lower in the
experiments compared to the controls, but overall the data
indicated that there was very little reduction of the type IV
collagenase levels over a 9-h interval. As an alternative ap
proach, following pulse labeling for 6 h, we measured collagen
ase turnover rates in cells exposed to serum-free medium con
taining 10 mg/ml PC-3 ML CM and 1.0 /¿M
taxol (Table 2).
The data showed that the cytoplasmic levels of ['H]collagenase
dropped from about 25,000 ±400 cpm to 15,000 ±300 cpm
during the first 3 h. Conversely, the ['H]collagenase levels in
collagenase
secreted40
the medium rose to about 12,000 ±cpm as a result of secretion
in the first 3 h. The levels of ['H]collagenase in the cytoplasm
±0.40
0.05 ±0.01
0.06 ±0.02
and medium remained fairly constant at levels comparable to
that measured at 3 h after 6- and 9-h intervals. We interpret
Table 4 Effects of taxol on PC-3 ML cell invasion
the data to mean that collagenase secretion probably occurs for
The cells were prelabeled with 200 >iCi/ml ['HJmethionine for 6 h, seeded at
the first 3 h of exposure to taxol but was then inhibited by 6
1x10' cells/well for 3 h, and then exposed to taxol (5-jil aliquots) for 1 to 9 h.
and 9 h as a result of the diffusion of taxol in the cells. We
Next, PC-3 M L conditioned medium ( 10 mg/ml protein) was added to the bottom
compartment for 6 h. Cells were harvested to determine the extent of invasion by
conclude that taxol probably has no direct effects on stability
scintillation counting (i.e., the percentage invasion is equal to the percentage of
and turnover of cytoplasmic collagenase. Also, the PC-3 ML
radioactivity in the bottom compartment versus the total radioactivity added per
CM does not appear to influence the stability of collagenase in
well). The data were averaged from triplicate experiments ±SD.
the taxol-treated cells.
timeTaxol(CM)0.00"0.010.050.100.501.001
Treatment
It was important to compare if total protein secretion or type
h8.4
h8.6
h8.0
IV collagenase secretion were inhibited by taxol in radiolabeled
±
0.27.9
±
0.47.8
±
±0.26.3 0.45.5
0.38.3
±
cells. Table 3 shows that the total labeled protein secreted in
±0.13.1 0.22.0
±
±0.16.2
0.36.0
±
0.23.0
±
±0.1000 response to PC-3 ML CM (10 mg/ml) was about 300 ±200
0.43.1
±
0.30.5
±
±0.30009h°8.3
0.23.1
±
±0.1006 ±0.1009h8.1
±0.10.4
cpm after 6 h and 200 ±95 cpm following 9 h preexposure of
±0.10.1
the cells to taxol, indicating inhibition of total secretion of
±0.23
protein. Control cells which were not treated with taxol secreted
" Taxol was removed from the medium for 18 h prior to beginning the invasion
total protein counts of ~360,000 ±200 cpm. Immunoprecipi
assay.
* Aliquots (5 ul) of castor oil were added.
tation measurements of the amounts of 'H-labeled type IV
nases (i.e., at M, 57,000) was also inhibited. The cells failed to
recover an ability to secrete collagenase following washes and
incubation in fresh medium in the absence of taxol for 48 h
(data not shown). Since the inhibition of secretion was coinci
dental with the disruption of the microtubules, we interpret the
data (Figs. 1-3) to mean that taxol induced rearrangements or
changes in the microtubules might interfere with their func
tional ability to mediate protease vesicle transport and secretion
of the gelatinases.
Effects of Taxol on Protein Synthesis, Turnover, and Secretion.
Collagenase secretion is thought to occur immediately following
synthesis (15). Therefore, we tested to see if taxol might indi
rectly block secretion by altering the half-life of type IV colla
genase or by inhibiting either total protein synthesis or type IV
collagenase synthesis. Table 2 summarizes the data from these
studies.
After pulse labeling for 6 h, immunoprecipitation
studies
collagenase secreted revealed that the medium contained 50 ±
10 cpm (after 6 h) and 60 ±20 cpm (after 9 h) exposure to
taxol, indicating an inhibition of protease secretion. By com
parison, the medium obtained from control cells contained
40,000 ±400 cpm. In sum, the data showed that taxol inhibited
both the synthesis and secretion of the M, 72,000 type IV
collagenase. We suggest that when type IV secretion is blocked,
translation is somehow inhibited, perhaps by mechanisms de
pendent on the cytoplasmic processing and packaging of the
protease. Interestingly, total protein secretion was also blocked
perhaps as a direct result of the disruption of the microtubule
distribution.
Boyden Chamber Invasion Studies. Boyden chemotactic as
says have been developed previously for studying the ability of
tumor cells to invade the basement membrane material (13, 15,
20). Utilizing this approach, we have quantitatively measured
the influence of taxol on the ability of the PC-3 ML cells to
penetrate Matrigel (Table 4). The measurable levels of invasion
3778
ANTIMETASTATIC
EFFECTS OF TAXOL ON PROSTATE TUMOR
observed in untreated cells (i.e., the ability to penetrate through
the Matrigel) was about 8 percent. Similar amounts of invasion
(about 7.9%) were observed in cells exposed to low taxol levels
(<0.01 MMfor 3 h) Invasion was partially inhibited at slightly
higher taxol levels (0.05 to 0.10 MM taxol for 1 to 6 h). In
comparison, the invasive response was reduced to zero in the
cells exposed to 0.1 to 1.0 MMtaxol for 3 to 9 h. The inhibitory
influence of taxol was not reversible in cells exposed to 0.1 to
1.0 MMtaxol for 9 h when taxol was subsequently removed from
the medium for 18 h. Taxol blockage of the secretion of
collagenases would play a significant role in preventing pene
tration of the Matrigel (14, 17).
Cell Attachment and Motility Studies. During invasion, the
cells must execute a series of complex maneuvers involving
partial detachment from the substrate, cell elongation and
movement through the matrix. We tested if taxol might indi
rectly block cell detachment/attachment
to substrate, and/or
interfere with cell motility. Relatively low levels of taxol (i.e.,
0.5 MMfor 3 h) were found to completely inhibit attachment of
the PC-3 ML subline (at 1 x IO6 cells/ml in suspension) to
Matrigel (2 mg/ml), type IV collagen (2 mg/ml)
substrates in vitro. Light microscopic studies of
stained filters (American Scientific Products, New
NJ) showed that 30-40 cells/cm- normally attached
and plastic
Diffi-QuikBrunswick,
after about
3 h, regardless of which substrate was used. Following exposure
to taxol (0.1 MMfor 3 h) only 1, 3 and 0 cells/cm2 attached to
the above substrates, respectively.
Taxol also blocked cell migration through membrane pores
(8 MM)in Boyden chemotactic chambers. Here, the membranes
were lightly coated with Matrigel (0.1 mg/ml) (so as not to
block the pores), in order to promote maximal cell attachment
and migration (15). PC-3 ML cells were then seeded at 1 x
106/ml in the top compartment, allowed to attach for 3 h, and
the unattached cells removed (i.e., «100 cells/ml) prior to
starting the experiment. Hemacytometer counts of cells col
lected from the bottom chamber with trypsin-EDTA showed
about 20% (i.e., 0.2 x IO6cells) normally migrated through the
pores by 3 h in response to PC-3 ML CM (10 mg/ml) added
to the bottom compartment. If the attached cells were exposed
to 0.5 MMtaxol for 3 h, washed and then stimulated to migrate,
a very small number of the cells (about 5 to 6 cells per well)
migrated to the bottom compartment by 3 and 6 h. Light
microscopy confirmed that the cells remained attached to the
membranes in the top compartment, indicating taxol interfered
with cell detachment. Northern blots showed that type IV
collagenase transcription was up regulated in response to the
PC-3 ML CM. Taxol (1.0 MM)alone did not turn on transcrip
tion and taxol pretreatment (0.1 to 1.0 MMfor 6 h) prior to
activation with PC-3 ML CM failed to inhibit upregulation of
transcription. We suggest that taxol's effects on microtubules
might prevent detachment and indirectly block cell motility, as
has been previously shown for fibroblasts (20). The combined
inhibitory activity of taxol on protease secretion, cell attach
ment/detachment
and motility, appear to completely block
invasion.
Activity of Taxol in Vivo: SCID Mouse Studies. We investi
gated if taxol inhibited the invasive metastatic activity of PC-3
ML cells in SCIDs. The PC-3 ML cells were exposed to taxol
(0.1 to 1.0 MM)for 0, 3 and 6 h, washed 3 x with DMEM and
injected i.v. in SCID mice (Table 5) according to methods
described previously (13). With untreated cells, the mice exhib
ited tumors in the lumbar vertebrae (greater than 80%) after 20
days following i.v. injection of 2 x IO5 cells/ml in 0.2 ml (13).
Table 5 Effects of taxol on PC-3 ML métastasesto the lumbar vertebrae
in SCIDs
Cells in suspension were exposed to taxol immediately prior to injection i.v.
in SCIDs. The cells were washed 3 times with Dulbecco's modified Eagle's
medium to remove unincorporated taxol, and 0.2 ml at 2 x 10' cells/ml as
injected per mouse. Mice were left for 20 days and the presence of tumors in the
lumbar vertebrae was assessed. Taxol was solubilized at 1 mg/ml in 50% polyoxyethylated castor oil (Cremophor EL) and 50% ethanol (USP), and aliquots were
added to Dulbecco's modified Eagle's medium. Due to toxicity, taxol and/or the
Cremophor vehicle were administered to mice in 0.05-ml dosages over 4-h periods
(0.2 ml total volume/mouse/day). To reduce acute hypersensitivity, mice received
i.v. injections of 1 mg dexamethasone at 2-h intervals, 3 times over 6 h prior to
administering the drug. One h prior to drug treatment, mice were given 2 mg
diphenhydramine and 10 mg Cimetidine i.v. in 0.05-ml volumes of doubledistilled H2O. Control mice were given the Cremophor vehicle. Mice were
carefully monitored and given oxygen if they exhibited hypersensitivity. Mice
were sacrificed by cervical dislocation if toxicity was apparent.
time3h4/20
Taxol(MM)0.1
0.5
1.0Oh18/20
17/20
18/20Treatment
0/20
0/206h1/20
0/20
0/20
When the cells were pre-exposed to taxol at 0.1 MMfor 3 h and
6 h, a substantially reduced number of the mice (4/20 and
1/20, respectively) exhibited métastasesto the vertebrae. Tu
mors were not present in other tissues (i.e., lung, liver, kidney,
spleen, brain) as determined by gross dissections and histology.
Fig. 4, is an H & E image of a tumor in the lumbar vertebrae
of a mouse injected with PC-3 ML cells previously exposed to
reduced levels of taxol, 0.1 MMtaxol for 3 h. The tumor size
and appearance (Fig. 4A) was not unlike that observed in mice
injected with untreated cells. Moreover, no obvious microtubule
bundling was apparent in the mitotic spindles following immunofluorescence labeling with beta tubulin antibodies (Fig.
4B). Therefore, we believe that taxol was not taken up by these
cells in significant amounts.
Preexposure of the cells to 0.5 or 1.0 MMtaxol for 3 to 6 h
prior to injection i.v. completely blocked métastases.Gross
dissections and histology revealed that none of the mice had
tumor tissue in the vertebrae or other tissues. We believe, that
this may result from poor survival, and/or an inability of the
cells to invade tissue and/or to grow tumors.
When 10 mice were injected with excess untreated cells (2 x
IO7 cells/ml in 0.2 ml) numerous lung nodules formed by 20
days (13). In mice injected with taxol treated cells (0.5 MMfor
6 h), absolutely no tumors were found after 20 days, 30 days
and 60 days, indicating cell number and time were probably not
limiting factors.
Effect of Taxol on Tumor Growth in Vivo. Previous experience
has shown that bone tumors are usually evident after 5 days
following the injection of PC-3 ML cells at 2 x 10* cells per
ml (13). To determine if taxol prevented tumor growth in vivo,
SCIDs were injected i.v. with PC-3 ML cells (2 x 10s cells/ml
in 0.2 ml) and left 5 days. On day 6, the mice were injected i.v.
via the tail vein with taxol (50 mg/rrr/day and 250 mg/m2/day
in 0.2 mis). Ten mice were treated with each taxol dosage tested
and five control mice received equivalent amounts of polyoxyethylated castor oil, the vehicle in which taxol was formulated.
After 15 days of treatment, the mice were sacrificed and ex
amined for tumors by dissection and histology. Gross dissection
revealed that tumors grew specifically in the lumbar vertebrae
(i.e., filling the bone marrow) in all the control mice. The bone
was usually destroyed in several areas and the tumors had
metastasized into the peritoneal cavity. Gross dissection of the
taxol treated mice showed that none of the 20 mice exhibited
noticeable tumors in any tissues examined (lungs, liver, colon,
3779
ANTIMETASTATIC
EFFECTS OF TAXOL ON PROSTATE TUMOR
Immunofluorescence labeling with beta-tubulin antibodies
revealed abnormal mitotic spindles (=15 pM in length) in tumor
cells of the taxol treated mice (Fig. 5). Also, the number of
mitotic cells detected per field of view were about 5 fold higher
than in the controls. Note that it was not technically possible
to resolve individual interphase microtubules (or bundles of
microtubules) as discrete structures, but the tubulin rich cyto
plasm fluoresced as a result of antibody binding to tubulin. The
control sections labeled with 2°ab-FITC alone failed to fluoresce. Interestingly, antibody labeling showed that the cells
were positive for the p 170 multi-drug resistance glycoprotein
(data not shown).
To determine if any tumor growth occurred months after the
taxol treatment was discontinued, mice were injected i.v. with
2 x 10s cells/ml (0.2 ml), left 5 days and exposed to taxol from
day 5 to 15 (i.e., 50 and 250 mg/nr/day). Treatment was
discontinued on day 15 and the mice sacrificed 3 months later.
Gross dissection and histology revealed that only 40 to 60% of
the mice contained tumors; 6/10 (50 mg/nr/day taxol) and
4/10 (250 mg/irr/day taxol). In all cases, the tumors observed
were small and delimited to the marrow cavity of the lumbar
vertebrae. Immunolabeling of cryosections with tubulin anti
bodies revealed that the spindles were about 1/3 shorter in
length (x5 /¿m)than normal spindle (=7 /¿m)found in control
cells. Taxol treatment may, therefore, have long-term inhibitory
effects on cell division and tumor growth. The apparent total
eradication of the vertebrae tumors, in at least 20 to 40 percent
of the mice tested, is noteworthy and may inadvertently arise
from cell death. Note that these latter values were corrected
(i.e., the raw data reduced 20%) to account for a potential 20%
failure by the PC-3 ML cells to initiate tumors (13).
The dosages tested here were somewhat low in comparison
to the levels which have been used in clinical trials (i.e., up to
350 mg/nr/day; 7-11). Unfortunately, if these higher dosages
were administered in mice, death or extreme hypersensitivity
(vomiting, droopiness) usually occurred in the animals.
In conclusion, the data demonstrates that taxol is of signifi
cant importance in the treatment of metastatic bone tumors,
including tumors exhibiting the multi-drug resistance phenotype (see Table I). More specifically, we believe that taxol has
therapeutic utility in inhibiting molecular processes activated
during invasion of the basement membrane and, therefore, may
be used to prevent micrometastases in malignant tissue during
surgery, radiation or chemotreatment. The future use of taxol
Fig. 4. A, transverse section of the lumbar vertebrae showing PC-3 ML cells
in the bone marrow. H & E, X1200. B, immunofluorescently ß-tubulin-labeled
PC-3 ML tumor tissue. Cells were exposed to 0.1 »JM
taxol for 6 h followed by
i.v. injection into mice; the mice were sacrificed at 20 days postinjection. x400.
testicles, muscle, brain, vertebrae). Histológica! studies revealed
that at day 20 there was tumor tissue but only in the bone
marrow of the lumbar vertebrae of the taxol treated mice. The
tumor burden in mice exposed to 50 mg/m2/day or 250 mg/
m:/day taxol was minimal and about the same as that observed
in untreated mice sacrificed at day 5.
Fig. 5. The tumor was established for 5 days and mice were exposed to taxol
(25 mg/m2/day) for 15 days. Shown are the normal spindle structures labeled
with /3-tubulin antibody. x600.
3780
ANTIMETASTATIC
EFFECTS OF TAXOL ON PROSTATE TUMOR
as an anti-cancer drug may be enhanced greatly with the devel
opment of non-toxic derivatives, or of receptor or antibody
mediated taxol delivery mechanisms to improve tumor uptake
and to reduce toxicity. Higher, more effective dosages could
then be used without generating severe long-term reactions by
patients.
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