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Experimental Therapeutics, Preclinical Pharmacology |
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 [S. Z., L. F., K. R. J., W. F.], and College of Pharmacy, Zhejiang University, Hangzhou 310027, China [S. Z., Y. Z. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Combination therapy with multiple drugs is a common practice in the treatment of cancer. The promising clinical activity of docetaxel has promoted considerable interest in combining this drug with other antitumor agents, such as etoposide, cyclophosphamide, 5-fluorouracil, and doxorubicin (4 , 9) . A number of these docetaxel-containing combinations are currently undergoing clinical evaluations, and preliminary results appear to be encouraging (9) . Doxorubicin, a derivative of anthracyclines, is one of the most active agents with a broad spectrum of activity against solid tumors and hematological malignancies (10) . The combination of docetaxel and doxorubicin has been proven effective in first-line treatment of metastatic breast cancer, with high response rates and acceptable toxicity (9 , 11) . In the present study, we conducted in vitro evaluations of the cytotoxic effects of docetaxel and doxorubicin against human breast and ovarian tumor cells in vitro. Our results demonstrated that pretreatment of tumor cell with doxorubicin or simultaneous exposure of tumor cell to doxorubicin could significantly repress the cell-killing activity as well as the general cytotoxic effect of docetaxel against tumor cells in vitro. These findings indicate that the interaction between docetaxel and doxorubicin is highly schedule dependent. The optimal schedule of this combination might be sequential exposure to docetaxel followed by doxorubicin.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determination of Internucleosomal DNA Cleavage.
Internucleosomal DNA fragmentation was assayed by a modification of
previously described methods (12
, 14)
. After treatment of
cells with various concentrations of docetaxel, doxorubicin, and their
combinations, cells were harvested, counted, and washed with PBS at
4°C. Cells were then suspended in lysis solution containing 20
mM Tris-HCl, 5 mM EDTA, and 5% (v/v) Triton
X-100 for 30 min on ice. The remaining steps for DNA fragmentation were
performed exactly as described previously (12)
. DNA
samples were analyzed by electrophoresis in a 1.5% agarose slab gel
containing 0.2 µg/ml ethidium bromide and visualized under UV
illumination.
Flow Cytometric Analysis.
Cell sample preparation and
PI3
staining were
performed according to the method described by Nicoletti et
al. (15)
. Briefly, cells treated with docetaxel,
doxorubicin, or their combinations were harvested by trypsinization.
After being washed twice with PBS, cells were fixed in 1% formaldehyde
in PBS on ice and then dehydrated in 70% ethanol in PBS. Approximately
1 h before flow cytometry analysis, RNase A (1 mg/ml) and PI (10
µg/ml) were added to each sample. Samples were incubated at room
temperature in complete darkness for 30 min. Cell cycle
distribution was determined using a Coulter Epics V instrument (Coulter
Corp.) with an argon laser and excitation at 488 nm. The results
were analyzed using Elite 4.0 software (Phoenix Flow System, San Diego,
CA).
MTT Assays.
BCap37 and OV2008 cells were harvested with trypsin and resuspended to
a final concentration of 2 x 104 cells/ml
in fresh medium with 10% FCS. Aliquots of 0.2 ml from each cell
suspension were distributed evenly into 96-well tissue culture plates
with lids (Falcon, Oxnard, CA). Designated columns were treated with 5
nM docetaxel, 100 nM doxorubicin, or their
combinations. One column from each plate contained medium alone, and
another column contained cells without drug as a blank control. After
cells were incubated at 37°C for 24 h, 0.1 ml of MTT (1 mg/ml)
solution was added to each well. After a 3-h incubation at 37°C to
allow viable cells to reduce the yellow MTT into dark blue Formosan
crystals, the resulting crystals were dissolved in 100 µl of DMSO.
The absorbance in individual wells was determined at 562 nm by a
microplate Reader (Molecular Devices). The fractional effect associated
with a range of concentrations was determined for each drug alone and
for various drug combinations. The data were analyzed by CalcuSyn
version 1.1 software (Biosft), assuming a mutually nonexclusive model.
The combination index was used to signify antagonism (combination
index > 1), additivity (combination index = 10), or
synergism (combination index < 1).
Morphological Examination through Cytospin Preparation.
Cells treated with docetaxel and/or doxorubicin were harvested by
trypsinization at the times indicated and washed twice with PBS. Cell
numbers were determined with a hemocytometer, and approximately
0.5–1 x 105 cells were plated onto
microscope slides using the Cytospin 3 cell preparation system
(Shandon, Pittsburgh, PA). Slides were air dried and fixed in acetone
before Wright-Giemsa staining and then examined using bright-field
microscopy.
Western Blotting.
Cells treated with docetaxel, doxorubicin, or their combinations at
different schedules were harvested by trypsinization after a 24-h
exposure. Protein extraction and immunoblot procedures were performed
as described previously (16)
. Briefly, protein samples
were loaded onto a 12% SDS polyacrylamide gel at equal protein
concentrations. After electrophoresis, samples were transferred to a
nitrocellulose membrane according to the Bio-Rad protocol. Primary
antibodies against p53, bcl-2 (Santa Cruz Biotechnology, Santa Cruz,
CA), c-raf, and p21 (Transduction Laboratories) were used at
1:1000 dilution (
0.3 µg/ml) in 3% BSA-PBS-T (PBS containing 0.5%
Tween 20). The secondary antibody, goat antimouse IgG conjugated to
horseradish peroxidase, was used at a concentration of 0.1 µg/ml in
3% BSA-PBS-T (Jackson ImmunoResearch). The immunoreactive bands were
visualized using a chemiluminescent substrate to horseradish peroxidase
(Amersham) and exposure to Kodak X-OMAT film.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the DNA fragmentation
after BCap37 cells were treated with different concentrations of
docetaxel (1–100 nM) for 48 h. The
characteristic DNA fragmentation ladder was observed in the cells
treated with 
5 nM docetaxel. This
indicates that docetaxel is about 10 times stronger than paclitaxel in
the induction of apoptotic cell death (12)
. In addition,
we also examined the cells treated with 5 nM
docetaxel over different time courses, and we determined that, at this
concentration, DNA fragmentation was detectable at 24 h of drug
exposure, but more fragmented DNA was observed by 48 h of drug
treatment (see Fig. 1B
). Next, flow cytometric assays were
performed to analyze the kinetic changes of cell cycle distribution and
apoptotic cell death in docetaxel-treated BCap37 cells. The results
depicted in Fig. 2
indicate that
docetaxel first causes cell cycle arrest at the
G2-M phase. This was observed 3 h after the
addition of docetaxel and continued to increase through 24 h of
incubation. Subsequently, a distinct peak representing an apoptotic
cell population (Fig. 2
, Ap) was detected. This
apoptotic peak continuously increased and became predominate
after 48 h of drug treatment. These results indicate that
docetaxel, similar to paclitaxel, can cause both mitotic arrest and
apoptotic cell death, and apoptotic events seem to occur after mitotic
arrest.
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. Next, the
sequential administration of these two drugs was examined by treatment
of tumor cells with 5 nM docetaxel, 100 nM
doxorubicin, or their combinations at various schedules for 24 and
48 h. The experimental results, which are summarized in Fig. 4
, show that pretreatment or simultaneous
treatment of tumor cells with 100 nM doxorubicin produced
less cytotoxic effects than treatment with docetaxel alone.
However, an increased overall cytotoxicity was observed when these
tumor cells were exposed to docetaxel for 12 h before the addition
of doxorubicin (Fig. 4)
. These results suggested that pretreatment or
simultaneous treatment with doxorubicin might antagonize the cytotoxic
effect of docetaxel. Furthermore, we performed the median effect
analysis with a fixed concentration ratio of 1:20
(docetaxel:doxorubicin). The fractional inhibition for each drug in
combination was analyzed in relation to the fractional inhibition
observed with each drug alone using CalcuSyn software with a mutually
nonexclusive model (see Fig. 5
). The
results indicated that simultaneous treatment or sequential doxorubicin
followed by docetaxel produced significant antagonistic effects.
However, when BCap37 cells were exposed to docetaxel for 12 h
before the addition of doxorubicin, the antagonistic effect gradually
disappeared. Instead, a marginal additive effect was observed with an
increase in drug concentrations. These findings indicate that
doxorubicin possesses schedule-dependent antagonism on the antitumor
activity of docetaxel in vitro.
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shows the DNA
fragmentation analyses of BCap37 and OV2008 cells exposed to docetaxel,
doxorubicin, or their combinations. As described above, the
characteristic DNA fragmentation ladders were observed in both BCap37
and OV2008 cells exposed to 5 nM docetaxel for 48 h,
but no DNA fragmentation was detected when these cells were treated
with 100 nM doxorubicin alone. When these cells were
exposed to both drugs simultaneously or pretreated with doxorubicin for
12 h, docetaxel-induced DNA fragmentation was dramatically
inhibited, particularly in the group pretreated with doxorubicin (see
Fig. 6
). However, when cells were exposed
to docetaxel first for 12 h, doxorubicin was unable to inhibit
docetaxel-induced DNA fragmentation (Fig. 6)
. Furthermore, the
inhibitory effect of doxorubicin on docetaxel-induced cell death was
investigated by flow cytometric analyses. From the results depicted in
Fig. 7
, we can clearly see that apoptotic
cell death induced by docetaxel (Fig. 7
, Ap) was
significantly repressed in the cells treated simultaneously or
pretreated with doxorubicin. These findings further confirmed that
doxorubicin could antagonize the cytotoxic effects of docetaxel against
solid tumor cells.
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). Furthermore, we counted those cells
that appeared to contain condensed chromosomes. The results summarized
in Table 1
indicate that more than 50%
of cells in the groups treated with docetaxel alone or pretreated with
docetaxel before doxorubicin were arrested at M phase after 48 h
of drug exposure. However, the percentage of mitotic cells was
dramatically decreased when cells were cotreated or pretreated with
doxorubicin. These results indicated that doxorubicin alone obviously
caused predominantly G2 arrest rather than
mitotic arrest. Pretreatment or simultaneous treatment of tumor cells
with doxorubicin prevented mitotic arrest caused by docetaxel (Fig. 8
;
Table 1
).
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indicate that docetaxel and doxorubicin
have little affect on either p21Waf1 or p53
protein, although a slight increase of p21Waf1
protein was observed in the cells treated with docetaxel alone.
However, docetaxel was found to induce phosphorylation of both bcl-2
and c-raf-1 (Fig. 9)
. Doxorubicin alone
did not alter either bcl-2 or c-raf-1 protein (Fig. 9
, Lane
3), but when BCap37 cells were exposed to docetaxel and
doxorubicin simultaneously or exposed to doxorubicin first for 12 h, the ability of docetaxel to induce bcl-2 or c-raf-1 phosphorylation
was completely inhibited (Fig. 6
, Lanes 4 and 6).
These findings suggest that doxorubicin can prevent docetaxel-induced
phosphorylation of bcl-2 and c-raf-1.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Docetaxel is a novel member of the taxane family. The promising
clinical activity of docetaxel has promoted considerable
interest in combining this drug with other antimitotic agents. In
clinical trials, doxorubicin is one of these agents used with docetaxel
in first-line treatment of advanced breast cancer and certain other
solid tumors (4
, 9)
. In this study, we investigated the
cytotoxic interaction between docetaxel and doxorubicin against human
breast and ovarian cancer cells in vitro. We observed that
simultaneous exposure to docetaxel and doxorubicin produced
antagonistic effects in these tumor cells. Sequential exposure of tumor
cells to doxorubicin followed by docetaxel also produced significant
antagonistic effects. However, no antagonistic effect was observed when
tumor cells were treated with docetaxel before being treated with
doxorubicin. In fact, the overall cytotoxicity produced by such a
sequential combination was even slightly higher than the cytotoxicity
produced by exposure to docetaxel alone (see Figs. 4
5
). These
findings suggest that simultaneous exposure or treatment with
doxorubicin before docetaxel is inadequate for the combination of these
two drugs. The optimal schedule for this combination may be the
sequential administration of docetaxel followed by doxorubicin.
To analyze the possible mechanism by which doxorubicin interferes with
the cytotoxic effects of docetaxel, we performed a series of
experiments including DNA fragmentation, flow cytometry, and cytospin
analyses. Our results demonstrated that doxorubicin affected the
cytotoxic effects of docetaxel on both mitotic arrest and apoptosis
(Figs. 6
7
8
; Table 1
). Docetaxel, like paclitaxel, is an antimitotic
agent. Antimitotic agents largely disrupt the dynamic tubulin network
in individual cells, resulting in aberrant aster formation. Thus,
affected cells are unable to transverse successfully from metaphase to
anaphase (1
, 2)
. Ultimately, the prolonged mitotic arrest
in most of these cells may lead to apoptotic cell death. For example,
paclitaxel has proven especially effective in its cell killing,
apparently through disruption of the microtubule network. By
morphological observation and flow cytometric analyses, we also noted
that the majority of tumor cells treated with docetaxel were arrested
at G2-M phase and subsequently underwent
apoptosis (Figs. 2
3
4
5
6
7)
. These results suggest a possible correlation
between mitotic arrest and apoptosis, although these data cannot
exclude the possibility that docetaxel, like paclitaxel, may also cause
apoptotic cell death via a signal pathway independent of mitotic arrest
(27)
. Indeed, our recent studies have demonstrated that
baccatin III, a precursor of paclitaxel and docetaxel, could cause
tumor cell death without mitotic arrest (28)
. In the
present study, doxorubicin was found to repress the cytotoxic effect of
docetaxel in both mitotic arrest and apoptosis unless docetaxel was
administered before doxorubicin. Interestingly, however, flow
cytometric assays showed that doxorubicin alone caused
G2-M-phase arrest (Fig. 7)
, although cytospin and
morphological examinations clearly indicated that doxorubicin did not
cause mitotic arrest in either BCap37 or OV2008 cells. Instead,
pretreatment with doxorubicin could significantly block
docetaxel-induced mitotic arrest (Fig. 8
; Table 1
). These results
implied that doxorubicin may actually cause predominantly
G2 arrest rather than mitotic arrest. In flow
cytometry, both G2 and mitotic arrest are
included in the G2-M-phase peak, and it is hard
to distinguish them (Fig. 7)
, but they can be easily distinguished by
cytospin assay and morphological examination. Thus, although the
G2-M-phase arrest by docetaxel was exclusively
mitotic, the G2-M-phase arrest by doxorubicin was
exclusively G2 arrest, which in turn inhibited
the mitotic arrest caused by docetaxel. A recent study conducted by
Blagosklonny et al. (29)
also reported that
doxorubicin caused predominantly G2 arrest and
prevented mitotic arrest induced by paclitaxel in HCT116 cells.
Therefore, it appears that the mechanism by which doxorubicin
antagonizes the cytotoxic effects of docetaxel is, in part, by
preventing the mitotic arrest of tumor cells.
Previous studies in several laboratories including ours have
demonstrated that paclitaxel and several other antimitotic agents could
induce bcl-2 hyperphosphorylation (17
, 30)
. Due to this
posttranslational modification, bcl-2 has been postulated to lose its
ability to block apoptosis (17)
. More recently, the
phosphorylation of bcl-2 has also been suggested to reflect the
damage of microtubules because the modification is commonly induced by
antimicrotubule agents and always occurs in the
G2-M phase of the cell cycle (21)
.
Moreover, Schandl et al. (31
, 32)
have reported
recently that this phosphorylation even occurs normally as part of
mitosis, suggesting that the phosphorylation of bcl-2 was more likely
an implication of G2-M-phase arrest. In this
study, we performed Western blot analysis and determined that docetaxel
can also induce bcl-2 phosphorylation. Interestingly, the combination
of docetaxel with doxorubicin blocked the phosphorylation of bcl-2.
Coincidentally, c-raf-1 phosphorylation was observed in BCap37 cells
after exposure to docetaxel (Fig. 9)
. c-raf-1 activation through
phosphorylation has been demonstrated after mitotic agent treatment and
has been linked to bcl-2 phosphorylation and the subsequent induction
of apoptosis (33)
. These findings may provide another
piece of evidence that doxorubicin antagonizes the cytotoxic effects of
docetaxel by preventing the mitotic arrest of tumor cells.
In summary, this study investigates the possible influence of doxorubicin on the cytotoxic effect of docetaxel against human breast and ovarian cancer cells in vitro. Our results demonstrate that antagonistic interaction occurred when tumor cells were exposed to two drugs simultaneously or exposed to doxorubicin before docetaxel. Further analyses demonstrated that doxorubicin could interfere with the cytotoxic effect of docetaxel on both mitotic arrest and apoptotic cell death. These findings suggest that doxorubicin might antagonize the cytotoxic effects of docetaxel against tumor cells by preventing tumor cells from progressing to the M phase of the cell cycle. On the other hand, the combination of doxorubicin and docetaxel is commonly used in the treatment of certain human solid tumors. Our results also suggest that careful consideration or further experimental evaluation using animal models might be necessary for the combination of these two drugs.
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FOOTNOTES |
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1 Supported by NIH Grants CA71851 and CA82440 (to
W. F.). 
2 To whom requests for reprints should be
addressed, at Department of Pathology and Laboratory Medicine, Medical
University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.
Phone: (843) 792-5108; Fax: (843) 792-7762; E-mail: fanw{at}musc.edu
3 The abbreviations used are: PI, propidium
iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide.
Received 12/ 3/99; revised 5/24/00; accepted 5/24/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, OV2008 cells; 
, BCap37 cells.
, control;
,
docetaxel; shaded box, doxorubicin; hatched
box, docetaxel + doxorubicin;
,
doxorubicin was added 12 h before docetaxel;
,
docetaxel was added 12 h before doxorubicin.
