docetaxel enhances tumor radioresponse in vivo1 · of the cell cycle most sensitive to ionizing...

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,

1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-

3263: Fax: (713) 794-5369.

INTRODUCTIONPaclitaxel and docetaxel are the prototypes of taxanes, a

new class of potent anticancer agents undergoing extensive

laboratory and clinical investigations (1-6). Both agents have

been shown to be cytotoxic in vitro for different tumor cell lines

(7-1 1), to exhibit antitumor activity in a variety of experimental

animal tumor systems ( 1 , 3), and to be effective in the treatment

of common cancers in humans (2, 4-6).

Both drugs are mitotic spindle poisons. They increase

tubulin polymerization, which promotes microtubule assembly,

and inhibit tubulin depolymerization, which stabilizes the mi-

crotubules (12-14). As a result, the cells are blocked in mitosis.

In addition to inducing mitotic arrest, taxanes induce cell death

by apoptosis in cell cultures (8, 15) and in in viva tumor systems

(1, 16). These studies mainly have been performed using pacli-

taxel. Although mitotically arrested cells are frequently destined

to die, they may also overcome the arrest and continue with

division (1, 16, 17). We observed recently that paclitaxel-

induced apoptosis, and not mitotic arrest, correlated with anti-

tumor efficacy of paclitaxel (1). A number of studies, particu-

larly those that used in vitro cell systems, demonstrated that

docetaxel was in general more cytotoxic than paclitaxel (10, 18,

19), and the increase was attributed to the higher affinity of

docetaxel for microtubules, its higher intracellular concentra-

tion, and its slower cellular efflux (19).

Because taxanes arrest cells in both G, and M, the phases

of the cell cycle most sensitive to ionizing radiation (20-22),

there have been several studies recently of the radiosensitizing

potential of these drugs (7, 9, 23, 24), most of which used

paclitaxel (7, 9, 23). They showed that radioresponse of most in

vitro cell lines was enhanced by pretreatment with the drug; the

enhancement factors ranged between 1 .5 and 1 .8 (7, 9, 23, 25).

The radiosensitization was higher in actively proliferating than

plateau phase cells (25), and in general, it was most pronounced

if radiation was delivered when paclitaxel-treated cells showed

significant G,-M block. There were, however, cell lines that

exhibited G,-M arrest but no enhanced cell radiosensitivity,

apparently depending on the length of taxane exposure, with

longer times being more effective (9). Thus, in vitro cell radio-

sensitization by taxanes depends on a number of factors includ-

ing cell line, proliferative state of cells, interval between radia-

tion and drug administration, drug concentration, and the length

of exposure of cells to the drug. It may well depend on which of

the taxanes is administered.

Compared to a relatively large amount of information

accumulated on the ability of taxanes to radiosensitize cells in

vitro, only a limited number of studies have addressed the

radiomodifying action of taxanes in vivo, and these were, to our

knowledge, confined to paclitaxel (26-31). Paclitaxel increased

tumor radioresponse to both single-dose (26-30) and fraction-

ated (3 1 ) irradiation. We reported recently that paclitaxel given

within 3 days before irradiation can enhance radioresponse of a

number of murine tumors; the drug enhanced the rate of tumor

cure, delayed the appearance of tumor recurrences, and delayed

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2432 Docetaxel and Tumor Radioresponse

the rate of tumor growth (26, 28-30). The degree of radiopo-

tentiation ranged from 1 .2 to nearly 2.0, and in tumors that

exhibited paclitaxei-induced apoptosis (mostly adenocarcino-

mas), the degree of radiopotentiation was greater when the time

interval between administration of paclitaxel and tumor irradi-

ation was increased.

A dominant mechanism of radiopotentiation in these tu-

mors was reoxygenation of hypoxic cells within the tumor,

which resulted when paclitaxel-killed tumor cells were removed

by apoptosis (28, 30). In contrast, the squamous cell carcinoma

(SCC-Vil) treated with paclitaxel histologically showed only

mitotic arrest, which peaked 6 h after drug administration, at

which time the radioenhancement was the highest (30). Thus,

the in vivo radioresponse of tumors was enhanced by paclitaxel

mainly through two mechanisms: mitotic arrest and tumor

reoxygenation.

To provide therapeutic benefit, a radioenhancing agent

must potentiate the radioresponse of the tumor more than that of

normal dose-limiting tissues. We observed that in contrast to a

strong enhancing activity of paclitaxel on tumor radioresponse,

the drug only minimally affected radiation injury to both acute

and late responding normal tissues when given up to 4 days

before radiation (27, 29). This implied that paclitaxel would

greatly increase the therapeutic ratio of radiotherapy.

To our knowledge, no information is available on the

ability of docetaxel to affect either tumor or normal tissue

radioresponse of in vivo animal models. Therefore, the current

study was designed to investigate whether docetaxel induces

mitotic arrest and apoptosis in the munne mammary carcinoma

MCA-4 and whether these cellular effects of docetaxel are

associated with an increase in tumor radioresponse. In addition,

the study investigated whether docetaxel modulates radiation

injury to mouse jejunal mucosa and whether the drug can

increase the therapeutic ratio of radiotherapy.

MATERIALS AND METHODS

Mice. C3Hf/Kam mice from our own specific pathogen-

free mouse colony were used at an age of 5 months. Mean body

weight was 30 ± 3 (SD) g. Mice were housed three to six per

cage, fed sterilized pelleted food (Agway, Inc., Syracuse, NY)

and sterile water ad !ibitum, and exposed to a 12-h light/dark

cycle. The experimental protocol was approved by the Institu-

tional Animal Care and Use Committee. Mice were maintained

in a fully accredited animal facility (American Association for

Accreditation of Laboratory Animal Care) and in accordance

with present regulations and standards of the United States

Department of Health and Human Services.

Docetaxel. Docetaxel was obtained from Rhone-Poulenc

Rorer (Vitry Sur Seine Cedex, France) as a pure crystalline

powder and stored at 4#{176}C.A stock solution of 50 mg/ml was

prepared in absolute ethanol and stored at -20#{176}Cfor the dura-

tion of experiments. Treatment solutions were prepared by mix-

ing 1 volume of the ethanolic stock solution, 1 volume of

polysorbate 80 (Sigma Chemical Co., St. Louis, MO), and 18

volumes of 5% glucose water. The iv. injection volume per

mouse was 0.4 ml or 33 mg/kg for a 30-g mouse. This dose of

docetaxel is roughly equivalent to the clinically used human

dose of 100 mg/m2. Treatment solutions were kept on ice and

injected within 10 mm of formulation.

Tumor. The syngeneic mammary carcinoma MCA-4

was used in its fourth isotransplant generation. Solitary tumors

were generated in the muscle of the right leg of the mouse by

inoculation of 5 X i0� viable tumor cells in suspension. Tumor

cell suspensions were prepared by mechanical disruption and

enzymatic digestion of nonnecrotic tumor tissue. The method

has been fully described previously (32).

Radiation. Mice bearing 8-mm (arithmetic mean diam-

eter) tumors in the right hind leg were locally irradiated, with

single doses of 9, 15, or 21 Gy using a ‘37cesium small animal

irradiator. Irradiations were given under ambient air breathing

conditions at a dose rate of 7 Gy/min. During irradiation, the

mice were mechanically immobilized (unanesthetized) on a jig,

and the tumor was centered in a 3-cm diameter circular field.

When docetaxel and radiation were combined, docetaxel was

given 9 or 48 h before tumor irradiation. Times were chosen to

coincide with the peak mitotic blockade induced by docetaxel (9

h), the return of the mitotic index to near baseline levels, and the

near completion of apoptotic cell loss (48 h).

Mice used for assaying the jejunal epithelial response to

treatment were irradiated whole body with single doses of

9.2-14.8 Gy 250 kV X-rays at a dose rate of 1 .62 Gy/min. The

measured RBE of gamma rays relative to X-rays for the radia-

tion sources used for these experiments is 0.88-0.95, depending

on the end point studied: tumor cure, 0.95; jejunal crypt stem

cell survival, 0.88. Mice were irradiated awake in groups of six

while loosely restrained in a well-ventilated Lucite box. When

docetaxel was combined with radiation treatment, it was given

3, 9, or 48 h before irradiation. These times were chosen to

correspond with the time of peak mitotic blockade in the jeju-

num (3 h), peak mitotic blockade in the MCA-4 tumor (9 h), and

the return of mitotic indices to near normal levels (48 h).

Histological Determination of Mitotic and Apoptotic

Indices. Groups of mice, either normal or tumor bearing, were

treated with 33 mgfkg docetaxel and sacrificed 1, 3, 6, 9, 12, 16,

48, and 72 h later. Either the 8-mm diameter MCA-4 tumor or

a 2-cm segment of jejunum was excised and fixed in 10%

neutral buffered formalin and then processed for routine histo-

logical examination. H&E-stained 4-�i.m sections were scored

microscopically at X400 as described previously (1, 16, 27, 33,

34). For tumor specimens, 100 cells in five random nonnecrotic

areas were scored as interphase, mitotic, or apoptotic (1 , 16, 33).

Mean apoptotic and mitotic indices were based on 500 cells

from each of three to five mice for a total of 1500-2500 cells

per tumor treatment group. A total of 34 mice were used for

mitotic and apoptotic indices of tumor-bearing mice treated with

docetaxel.

For jejunal mucosa, 100 epithelial cells in complete longi-

tudinal crypt sections from five random areas of jejunal trans-

verse sections were scored as interphase, mitotic, or apoptotic

using morphological criteria established previously (27, 34).

Mean apoptotic and mitotic indices per treatment group were

based on 1500-8500 crypt cells from 3 to 17 mice per group. A

total of 70 mice were used for the time course of jejunal

response to docetaxel treatment.

Tumor Growth Delay. The antitumor effect of do-

cetaxel was determined by its ability to delay tumor growth



Clinical Cancer Research 2433

Table I Effect of docetaxel (DOC) on ra dioresponse of MCA-4 cells me asured by tumor growth delay

Tumor Growth Delay

Time in days

Treatment”

required to grow

from 8-12 mm

Absolute growth

delay”

Normalized

growth delay�

Enhancement

factors�’

No treatment 4.9 ± 0.4�

DOC 11.8±1.0

Radiation 9 Gy 8.8 ± 0.7 3.9 ± 0.7

DOC + 9 Gy (9 h) 18.2 ± 1.2 13.3 ± 1.2 6.4 ± 1.2 1.64

DOC + 9 Gy (48 h) 22.1 ± 1.! 17.2 ± 1.1 10.3 ± 1.1 2.64

Radiation 15 Gy 1 1 .9 ± 0.9 7.0 ± 0.9

DOC + 15 Gy (9 h) 22.9 ± 1.5 18.0 ± 1.5 11.1 ± 1.5 1.59

DOC + 15 Gy (48 h) 24.8 ± 1.3 19.9 ± 1.3 13.0 ± 1.3 1.86

Radiation 21 Gy 15.2 ± 1.3 10.3 ± 1.3

DOC + 21 Gy (9 h) 25.9 ± 2.2 21.0 ± 2.2 14.1 ± 2.2 1.37DOC + 21 Gy (48 h) 29.7 ± 1.2 24.9 ± 1.3 17.9 ± 1.2 1.74

(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

controls; docetaxel 3 h, 161 ; docetaxel 9 h, 159; and docetaxel

48 h, 162) to account for multiplicity of surviving cryptogenic

cells per crypt (35). Survival curves were fitted to the data using

least squares regression analysis.

RESULTSEnhancement of Tumor Radioresponse. Mice bearing

8-mm MCA-4 tumors were treated with docetaxel, 33 mg/kg

given iv. , to determine the effect of the drug on tumor growth

and its ability to induce mitotic arrest and apoptosis in tumor

cells. The drug strongly inhibited tumor growth, prolonging the

time tumors required to grow from 8 to 12 mm from 4.9 ± 0.4

days in control mice to 1 1 .8 ± 1.0 days (Table 1). Tumors from

untreated mice or mice treated with docetaxel 1, 3, 6, 9, 12, 16,

24, 48, or 72 h earlier were removed and histologically analyzed

for the presence of mitotically arrested and apoptotic cells. The

results, plotted in Fig. 1, show that docetaxel arrested cells in

mitosis by 3 h after treatment. The percentage of arrested cells

increased rapidly with time, achieving its peak of 22. 1 ± 2.8%

at 9 h after docetaxel. Following the peak, the percentage of

arrested mitoses declined, returning to background by 72 h after

treatment. Docetaxel also induced apoptosis, which began to

increase 6 h after its administration, and reached the peak of

10.3 ± 1 .6% at 24 h. The percentage then declined to the control

value at 72 h after docetaxel.

Mitotically arrested cells were observed throughout tumor

lobules, although they were more frequent at the periphery,

closer to blood vessels. Apoptotic cells were more evenly dis-

tributed throughout the lobules, although they also showed a

tendency to be located more at the periphery. These distribution

characteristics and the morphology of the arrested cells, many

having a characteristic “wagon wheel” appearance in which

chromosomes were arranged in a concentric zone beneath the

plasma membrane (Fig. 2B), were similar to those observed

after paclitaxel treatment (16).

Two additional histological features were noted in the

treated tumors. A large proportion of mitotically arrested cells

were disrupted, with nuclear material spilling into the extracel-

lular space (Fig. 2C). This nonapoptotic mode of cell death was

also observed in tumors treated with paclitaxel (16), but there it

was not as extensive. Although this cellular lysis could not be



0 6121824 48 72

HOURS AFTER DOCETAXEL


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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

radioenhancement factors, normalized tumor growth delays

were determined in the combined treatment groups and then

divided by the absolute tumor growth delays produced by cor-

responding radiation doses only. The factors obtained ranged

from 1.37 to 2.64.

To define the degree of docetaxel-induced enhancement of

tumor radioresponse more accurately, normalized tumor growth

delays were plotted as a function of radiation dose (Fig. 3).

Docetaxel treatment displaced the radiation dose-response

curves to lower radiation doses more for the 48-h than the 9-h

treatment interval, indicating that tumor radioresponse was en-

hanced. The enhancement factors were determined at isoeffec-

live radiation doses that resulted in tumor growth delay of 10

days, and they were 1.45 for docetaxel given 9 h and 2.33 for

docetaxel given 48 h before radiation. Docetaxel exhibited a

strong ability to enhance radioresponse of MCA-4 tumor. This

enhancement was more profound than that previously demon-

strated for paclitaxel (enhancement factors: 9 h, 1.19; 48 h, 1.86)

using the same tumor and similar experimental conditions (28).

Modulation of Normal Tissue Radioresponse. Before

testing whether docetaxel modulates radiation-inflicted injury to

jejunal mucosa, we determined whether the drug induces mitotic

arrest and apoptosis in this tissue and the kinetics of these

cellular effects. Mice were treated with docetaxel, 33 mg/kg iv.,

and sacrificed at the same times after treatment as was done for

tumor studies, and their jejunums were removed and prepared

for histological analyses. The results, presented in Fig. 1B, show

that docetaxel was effective in inducing both mitotic arrest and

apoptosis in jejunum. The induction of mitotic arrest was more

rapid than that in MCA-4 tumor; the peak of 19.0 ± 0.4%

occurred 3 h after treatment. The decline was also rapid, reach-

ing background levels 9 h after treatment. Docetaxel was also

effective in inducing apoptosis in jejunal cells. which started to

be visible at 6 h after docetaxel administration and reached its

peak of about 19% between 9 and 12 h after treatment. The

percentage then rapidly declined, reaching a level slightly above

background at 24 h after docetaxel administration. It should be

noted that lytic disruption of mitotically arrested cells was less

frequently seen in jejunal mucosal cells after docetaxel treat-

ment than in the MCA-4 tumor.

To determine the effect of docetaxel on radioresponse of

jejunal mucosa, 33 mg/kg docetaxel was given iv., and then 3 h

(peak of mitotic arrest), 9 h (peak of apoptosis), or 48 h (when




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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



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Fig. 4 Radiation dose survival curves of mouse jejunal crypt cells at

3 h (A), 9 h (U), and 48 h ( #{149}) after injection of 33 mg/kg docetaxel or

those treated with radiation only (0). Each data point represents the

mean cell survival of 6 or 12 mice: bars, SE.

I I I I I I

8 10 12 14 16 18 20 22 8 9 10 11 12 13 14 15

RADIATiON DOSE (GY)


RADIATION DOSE (GY)

Fig. 3 Effect of docetaxel on MCA-4 tumor growth delay as a function

of radiation dose. Mice bearing 8-mm tumors in the right hind leg were

given 9. 15, or 21 Gy local tumor irradiation (0) or iv. injection of

docetaxel (33 mg/kg) followed 9 h (#{149})or 48 h (V) later by the samedoses of radiation. Groups consisted of seven or eight mice each. Bars.

SE. Data were fitted using linear regression.

our studies on the antitumor activity of paclitaxel and combi-

nation of paclitaxel and radiation ( 1 6, 26, 28). The present study

shows docetaxel alone was highly effective in slowing the

growth of this tumor, and at the cellular level, it induced marked

mitotic arrest and apoptosis. Both the extent and kinetics of

mitotic arrest and apoptosis (Fig. 1 ) were similar to that induced

by paclitaxel (16). The peak of mitotic arrest was 9 h. and of

apoptosis, 24 h after both agents. However, the dose of do-

cetaxel (33 mg/kg) that produced this effect was smaller than the

dose of paclitaxel (40 mg/kg) used in our earlier dose-response

studies (1, 28-30), which suggests that docetaxel is more effec-

tive than paclitaxel. A number of in vitro studies have shown

that on a concentration basis, docetaxel is more cytotoxic than

paclitaxel (10, 18), which was attributed to higher affinity of

docetaxel for microtubules, higher achievable intracellular con-

centration, and the slower cellular efflux of docetaxel (19).

Interestingly, histological analysis of MCA-4 tumors treated

with docetaxel (Fig. 2C) showed that both cell lysis and apop-

tosis were pronounced modes of cell death, whereas our earlier

studies showed that apoptosis was the dominant mode of cell

death in tumors treated with paclitaxel (1. 16).

To combine docetaxel with radiation on a biologically

rational basis, it was necessary to establish the kinetics of

mitotic arrest and apoptosis. The arrest of cells in mitosis is

considered to be the basis of cell radiosensitization by taxanes,

because mitotic cells are highly sensitive to radiation; the results

of most in vitro studies with paclitaxel support this rationale (7,

9, 23-25). However, our studies on the in vivo radiopotentiating

effects of paclitaxel in several murine tumors revealed two

major mechanisms: mitotic arrest and tumor reoxygenation.

Mitotic arrest was a dominant mechanism in SCC-VII, a tumor

that after treatment with paclitaxel exhibited mitotic arrest but

not apoptosis (30). However, in tumors that display both mitotic

arrest and apoptosis after paclitaxel. tumor reoxygenation was

the dominant mechanism ofradiosensitization (28, 30), although

the contribution of mitotic arrest was still observable (30).

Radiation was delivered 9 h after docetaxel to coincide

with the peak of tumor cell mitotic arrest or 48 h after docetaxel

to coincide with the time when a large proportion of tumor cells

was lost by apoptosis or cell lysis. At both time points, tumor

radioresponse was enhanced, but the enhancement was much

higher at the 48-h time point, when the enhancement factor was

2.33, than for the 9-h time point. when the enhancement factor

was 1 .45 (Fig. 3). Because the highest degree of enhancement

did not occur at the peak of mitotic arrest. the accumulation of

cells in mitosis cannot be considered the primary mechanism of

radioenhancement in this tumor. Some other mechanism(s).

most likely tumor reoxygenation due to massive cell loss, is

responsible. The results are similar to those observed earlier for

the same tumor treated with paclitaxel where tumor reoxygen-

ation was the dominant mechanism (28). In that study, the

enhancement was greatly reduced when tumors were irradiated

under hypoxic conditions, a procedure that makes all tumor cells

hypoxic. Direct measurement of tumor P#{176}2confirmed that

tumor reoxygenation did occur following treatment with pacli-

taxel (28). It was reported recently by Griffon-Etienne et a!. (36)

that docetaxel improved the oxygenation of MCA-4 carcinoma

and that it was more effective than paclitaxel. It should be noted

that the degree of radioenhancement induced by docetaxel (Fig.

3) was higher than that produced by paclitaxel in the same (26,

28) or other tumors (29, 30). Whether this can be attributed to

better tumor reoxygenation by docetaxel (36) or to other radio-




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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1997;3:2431-2438. Clin Cancer Res K A Mason, N R Hunter, M Milas, et al. Docetaxel enhances tumor radioresponse in vivo.

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