Docetaxel Enhances Tumor Radioresponse in Vivo1 · of the cell cycle most sensitive to ionizing radiation (20-22), there have been several studies recently of the radiosensitizing
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Vol. 3, 2431-2438, December 1997 Clinical Cancer Research 2431
Docetaxel Enhances Tumor Radioresponse in Vivo1
Kathryn A. Mason, Nancy R. Hunter, Mira Milas,
James L. Abbruzzese, and Luka Milas2
Departments of Experimental Radiation Oncology [K. A. M.,
N. R. H.. L. M.]. Surgical Oncology [M. M.J, and GastrointestinalOncology and Digestive Diseases IJ. L. Al, The University of TexasM. D. Anderson Cancer Center. Houston. Texas 77030
ABSTRACTAlthough the radiosensitizing potential of paclitaxel has
been investigated extensively in cancer treatment, a sister
taxane, docetaxel, has been studied rarely. We investigated
the ability of docetaxel to enhance in vivo tumor radiore-
sponse and influence radiation injury to normal tissue. In
addition, mitotic arrest and apoptosis in tumors and normal
tissues were assessed after docetaxel administration to de.
termine whether these cellular effects underly its radio-
modifying action. Mice bearing in their legs 8-mm isotrans-
plants of a murine mammary carcinoma, designated
MCA-4, were treated with 33 mg/kg docetaxel i.v., 9-21 Gy
single-dose local tumor irradiation, or both (in which case
radiation was given 9 or 48 h after docetaxel). Tumor
growth delay was the end point of the treatments. Mitotic
arrest and apoptosis were assayed 1-72 h after treatment
with docetaxel. Normal tissue radioresponse was determined
using jejunal crypt cell survival 3.5 days after mice were
exposed to 9.2-14.8 Gy single-dose, total-body irradiation;
the mice were treated with 33 mg/kg docetaxel i.v. 3, 9, or
48 h before irradiation. Docetaxel was assessed for its ability
to induce mitotic arrest and apoptosis in jejunum 1-72 h
after treatment. Docetaxel induced both mitotic arrest and
apoptosis in both tumor and jejunum. Mitotic arrest pre-
ceded apoptosis and peaked in the tumor at 9-12 h after
treatment; it peaked at 3 h in jejunum. Docetaxel enhanced
tumor radioresponse by a factor of 1.45 when the drug was
given 9 h before radiation and 2.33 when it was given 48 h
before. In contrast, it only slightly enhanced radiation-
induced damage of the jejunum and only when given 3 or 9 h
before irradiation. Thus, docetaxel given within 2 days be-
fore irradiation acted as a potent enhancer of tumor radio-
response and increased the therapeutic gain of irradiation.
Received 6/9/97: revised 8/25/97: accepted 9/1 1/97.
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.I Supported by Rhone-Poulenc Rorer Pharmaceuticals, Inc.
2 To whom requests for reprints should be addressed. at Department of
Experimental Radiation Oncology. M. D. Anderson Cancer Center,
(1 Mice bearing 8-mm tumors in the right hind leg were given iv. 33 mg/kg docetaxel (DOC) or local tumor irradiation. When the two agentswere combined, irradiation was given 9 or 48 h after docetaxel. Groups consisted of seven or eight mice each.
b Absolute tumor growth delay caused by radiation, docetaxel, or both agents is defined as the time in days tumors required to reach 1 2 mm from
the time of treatment initiation minus the time in days untreated tumors required to grow from 8 to I 2 mm.
( Normalized tumor growth delay is defined as the time in days for tumors to reach 1 2 mm in mice treated by the combination of docetaxel and
radiation minus the time in days to reach 12 mm in mice treated by docetaxel only.
‘I Enhancement factors: obtained by dividing normalized tumor growth delay in mice treated by docetaxel plus radiation by the absolute growth
delay in mice treated with radiation only.e Mean ± SE.
from 8 to 12 mm mean tumor diameter. Palpable tumors were
measured daily in three orthogonal directions with Vernier
calipers. Mice whose tumors had grown to 8-mm diameter were
randomly assigned to treatment groups: no treatment, radiation
only, docetaxel only, or docetaxel plus radiation. Tumors were
locally irradiated with 9, 15, or 21 Gy or injected iv. with 33
mg/kg docetaxel followed 9 or 48 h later by the same radiation
doses. Treatment groups consisted of 7-8 mice each for a total
of 86 mice evaluated for tumor growth delay.
Jejunal Crypt Survival. The survival of jejunal crypts
treated with radiation alone or with combined radiation/do-
cetaxel (33 mg/kg) given 3, 9, or 48 h previously was quantified
using the microcolony assay of Withers and Elkind (35). Groups
of 6 or 12 mice were given radiation doses over the dose range
of 9.2-14.8 Gy. At 3.5 days after irradiation, mice were sacri-
ficed by CO2 inhalation, and a 2-cm length of jejunum was
removed. Following fixation in 10% neutral buffered formalin,
four transverse tissue sections per mouse were cut at a thickness
of 4 �im and stained with H&E.
Tissue sections were scored microscopically at X 100. The
number of surviving crypts per circumference of jejunum was
scored for four tissue sections per mouse and averaged. The
number of surviving crypts per circumference was transformed
to surviving cells per circumference by applying a Poisson
correction based on the number of crypts at risk ( 16 1 in normal
Fig. 1 Mitotic arrest and apoptosis in MCA-4 tumor and in jejunal
mucosa treated with docetaxel. Mice bearing 8-mm-diameter MCA-4tumors in the right hind leg were treated with docetaxel (33 mg/kg iv.).
Groups ofthree to five mice were sacrificed I, 3, 6, 9, 12, 16, 24, 48, and72 h later. Tumors or jejunum were surgically removed and fixed inneutral buffered formalin prior to routine histological processing. H&E-
stained tissue sections were scored microscopically at X400. A total of500 cells/mouse were scored as interphase. mitotic, or apoptotic for bothMCA-4 and jejunum. #{149}.mitotic index; U, apoptotic index. Bars, SE.
quantified, the extensiveness of the process may have contrib-
uted to tumor cell death after docetaxel as much as or more than
apoptosis. The other notable histological feature was massive
tumor infiltration by mononuclear lymphoid cells noted at 48 h
and being particularly evident 72 h after treatment with do-
cetaxel (Fig. 2D). In some tumors, the infiltration was so cx-
tensive that individual tumor cells were scarce. The infiltration
of MCA-4 tumor with lymphoid cells was not seen after treat-
ment with paclitaxel (16).
To investigate whether and to what extent docetaxel en-
hances the radioresponse of MCA-4 and whether the magnitude
of radiopotentiation depends on the length of time between
docetaxel administration and tumor irradiation, mice bearing
8-mm tumors were given 33 mg/kg iv. docetaxel. and 9 or 48 h
later their tumors were locally irradiated with 9. 15, or 21 Gy
single doses of radiation. Thus, the radiation was given either at
the peak (9-h interval point) of mitotically arrested cells or when
the arrested cells had almost totally disappeared due to apoptosis
or cell lysis (48-h interval point). Tumor growth delay, i.e., time
in days tumors needed to grow from 8 to I 2 mm in diameter.
was used as the treatment end point (Table 1 ). Both docetaxel
and all radiation doses were strongly effective as single treat-
ments, but when combined. they produced tumor growth delays
longer than the additive effects of individual treatments, mdi-
eating that docetaxel enhanced tumor radioresponse. The degree
of enhancement depended on the time interval between do-
cetaxel administration and radiation delivery; it was higher
when the interval was 48 h than when it was 9 h. To obtain
Fig. 2 Histological appearance of MCA-4 tumors untreated (A) or treated with 33 mg/kg docetaxel (B-D). X 1000. A: short arrows, mitotic figures;long arrows, apoptotic cells. B, mitotically arrested cells showing characteristic coronal appearance of condensed chromatin (short arrow) andapoptotic (long arrow) cells 12 h after docetaxel. C. extensive cell disruption with numerous condensed chromosomes in intercellular spaces 24 h afterdocetaxel. D, tumor infiltration with mononuclear lymphoid cells 72 h after docetaxel.
both mitotic arrest and apoptosis normalized) later, the mice
were exposed to graded single, total-body doses of irradiation
ranging from 9.2 to 14.8 Gy. Controls were mice exposed to
radiation only. The effect of treatments expressed as surviving
cells per circumference of jejunum 3.5 days after irradiation is
plotted in Fig. 4. In all groups, radiation caused dose-dependent
reductions in the survival of crypt epithelial cells. Radiation
response curves at the peak of mitotic arrest (3 h after docetaxel)
and at the peak of apoptosis (9 h after docetaxel) were shifted to
the left to lower radiation doses, indicating an increased cellular
response to the combined treatment. The enhancement factors at
20 surviving cells isoeffective radiation dose were 1 .08 and 1.14
for 3 and 9 h, respectively. On the other hand, docetaxel ad-
ministered 48 h before radiation was slightly radioprotective.
DISCUSSION
The combination of chemotherapy and radiotherapy is in-
creasingly used in cancer therapy, particularly when the drugs
possess radiosensitizing properties. Such drugs reduce the num-
ber of clonogenic cells in tumors undergoing radiotherapy by
their own cytotoxic action and by rendering tumor cells more
susceptible to killing by ionizing radiation. An additional benefit
of the combined treatment is that chemotherapeutic drugs, by
virtue of their systemic activity, may act on metastatic disease
outside the radiation fields. Because of their strong cytotoxicity
and their ability to radiosensitize cells, ta.xanes have a high
potential to be effective in combination with radiotherapy. As
elaborated in the “Introduction,” there have been many studies
recently assessing the radiosensitizing potential of taxanes, but
the preclinical research was almost exclusively confined to
paclitaxel. Thus, very little is known about the interactions of
docetaxel with radiation, particularly for tumor and normal
tissue treatment in vivo.
A number of important issues relevant to the therapeutic
application of docetaxel when combined with radiation were
addressed by the experiments described in this study. These
include whether docetaxel enhances tumor radioresponse, what
cellular changes underlie the effect, and whether radioenhance-
ment depends on the time interval between docetaxel adminis-
tration and radiation delivery. In addition, we tested whether the
combination of docetaxel and radiation influences the response
of normal tissue to radiation, which is essential for the assess-
ment of whether docetaxel can increase the therapeutic ratio of
radiotherapy.
To test the tumor radiopotentiating ability of docetaxel, we
used adenocarcinoma MCA-4, a murine tumor used in most of
3 The abbreviation used is: TNF, tumor necrosis factor.
potentiation mechanisms is not yet clear. A mechanism that may
also be involved in docetaxel-induced radiopotentiation is the
toxicity of docetaxel against S-phase cells (37), a cell cycle
phase most resistant to ionizing radiation.
An interesting histological change in tumors treated with
docetaxel was massive infiltration with mononuclear cells (Fig.
2D), a phenomenon not observed after treatment with paclitaxel
(16). The cause of the mononuclear infiltration is unclear but
resembles that resulting from antitumor immunological reaction
(38). Paclitaxel was reported to be able to induce cytokine gene
expression. such as TNF3-a (39-41) and interleukin 1 (40), as
well as the release of TNF by macrophages (39). However,
docetaxel was unable to induce TNF-ct gene expression (41).
Research on this aspect of biological activities of taxanes is
scarce, but observations with paclitaxel suggest that taxanes
may influence production of cytokines that could then result in
tumor infiltration with mononuclear lymphoid cells. If the ob-
served infiltration of MCA-4 tumor with mononuclear cells
represents an antitumor rejection response, it could have con-
tributed as a mechanism to the antitumor efficacy of docetaxel-
only treatment and to its potentiation of tumor radioresponse.
Our earlier studies showed that elicitation or augmentation of
antitumor immune responses can greatly enhance the radiore-
sponse of murine tumors (38, 42, 43).
To be therapeutically beneficial. docetaxel or any other
radiopotentiating agent must increase tumor radioresponse more
than the radioresponse of the normal tissues that limit radiother-
apy. We tested the effect of docetaxel on damage to jejunal
mucosal cells. an acute radiation injury, and found 33 mg/kg
docetaxel equitoxic to 40 mg/kg paclitaxel (27. 29). The cells
responded to docetaxel treatment alone by more rapid mitotic
arrest and apoptosis than MCA-4 tumor cells. Mitotic arrest
peaked at 3 h, and apoptosis peaked between 9 and 1 2 h after
docetaxel. Also, the duration of these cellular changes, espe-
cially apoptosis, was much shorter in jejunum than in the tumor.
The reasons for the differences in response to docetaxel between
jejunal mucosa and MCA-4 tumor are not known but may be
related to more rapid cell proliferation in jejunum and more
rapid removal of docetaxel from mucosal epithelial cells. Sup-
porting this observation is the finding that docetaxel is elimi-
nated at a slower rate from tumors than from normal tissues
(3. 19).
The combination of docetaxel with radiation resulted in
more serious damage to jejunal mucosa compared to radiation-
only treatment (Fig. 4). when the drug was given at 3 h (peak of
mitotic arrest) or 9 h (peak of apoptosis) after docetaxel. when
the enhancement factors were 1.08 and 1.14. respectively. As in
the tumor, the highest increase in damage did not occur at the
peak of mitotic arrest. It is unclear whether the observed in-
crease in radiation injury was a true potentiation of radiation
response or whether it represented the sum of damages inflicted
by individual agents (27, 29). In either case. however, a thera-
peutic gain was achieved because the potentiation of tumor
radioresponse was greater than the potentiation ofjejunal crypt
injury at all treatment time intervals. This was particularly true
when docetaxel preceded radiation by 48 h because at this time,
tumor radiopotentiation was the greatest (2.33 enhancement
factor), when some normal tissue radioprotection was evident.
Overall, the results show that docetaxel is a strong poten-
tiator of radiation response of a murine adenocarcinoma when
given within 2 days before radiation. The enhancement was
more pronounced when docetaxel was administered 48 h rather
than 9 h before irradiation. a difference related to the kinetics of
docetaxel-induced mitotic arrest. apoptosis, and cell lysis. At
doses of 33 mg/kg docetaxel and 40 mg/kg paclitaxel, the
magnitude of docetaxel-induced tumor radiopotentiation was in
general greater than that which we reported earlier for paclitaxel
(26, 28-30), although normal tissue toxicity was equivalent (27,
29). Docetaxel modestly increased the radiation damage of
mouse jejunum when given within 9 h before irradiation but had
no deleterious effect when given 48 h before irradiation. There-
fore, docetaxel was able to increase therapeutic gain when
combined with radiotherapy in preclinical tumor and normal
tissue systems. thus demonstrating that it has a high potential to
be a successful potentiator of radiotherapy in the clinic.
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