Vol. 9, 4735– 4742, October 15, 2003
Clinical Cancer Research 4735
ZD1839 (Iressa) Modifies the Activity of Key Enzymes Linked to
Fluoropyrimidine Activity: Rational Basis for a New
Combination Therapy with Capecitabine
Nicolas Magné, Jean-Louis Fischel,
Alain Dubreuil, Patricia Formento,
Joseph Ciccolini, Jean-Louis Formento,
Céline Tiffon, Nicole Renée, Sandrine Marchetti,
Marie-Christine Etienne, and Gérard Milano1
Oncopharmacology Unit, Centre Antoine Lacassagne, 06189 Nice
Cedex 2 [N. M., J-L. Fi., A. D., P. F., J-L. F., C. T., N. R., S. M.,
M-C. E., G. M.], and Pharmacokinetics Laboratory, School of
Pharmacy, 13385 Marseille Cedex 05 [J. C.], France
creased cellular production of 5-fluorouracil anabolites
from 5ⴕ-DFUR when cells were preincubated with ZD1839.
Conclusions: These data demonstrate a strong synergistic interaction between ZD1839 and 5ⴕ-DFUR when ZD1839
is applied before or concurrently with 5ⴕ-DFUR. Such a
drug combination would have two advantages: (a) the theoretical advantage of tumor selectivity of epidermal growth
factor receptor-targeted therapy; and (b) the practical advantage of a combination therapy that could be administered p.o.
ABSTRACT
INTRODUCTION
Purpose: The efficacy of new oral fluoropyrimidines,
including capecitabine, is improved in cells expressing high
levels of thymidine phosphorylase (TP) and low levels of
thymidylate synthase (TS) and dihydropyrimidine dehydrogenase. We used a human head and neck cancer cell line
(CAL33) to examine the influence of cell cycle modifications
on TS, TP, and dihydropyrimidine dehydrogenase activity.
Experimental Design: Cells were exposed to the epidermal growth factor receptor tyrosine kinase inhibitor
ZD1839 (Iressa2) and 5ⴕ-deoxy-5-fluorouridine (5ⴕ-DFUR),
alone and in combination, for up to 96 h, and modifications
in cell cycle, enzyme activity, and gene expression were
examined.
Results: ZD1839 (24- to 72-h exposure) markedly reduced proliferation and caused a rapid increase in G0-G1
and a decrease in S phase; a 40-fold decrease in TS activity
at 24 h and a 2.5-fold increase in TP activity at 48 h were
observed. A significant link between TP activity and expression was observed (r2 ⴝ 0.98; P ⴝ 0.0068). Additional investigations pointed out an increased cellular production of
5-fluorouracil anabolites from 5ⴕ-DFUR when cells were
preincubated with ZD1839. Dose-effect curves of ZD1839
and 5ⴕ-DFUR, alone and in combination, were examined.
Combination indices for ZD1839 ⴙ 5ⴕ-DFUR were 0.58 ⴞ
0.1 and 0.63 ⴞ 0.1 for 50% survival and 25% survival,
respectively. Additional investigations pointed out an in-
Several recent clinical studies have shown that the rationally designed, tumor-selective, p.o.-administered fluoropyrimidine derivative capecitabine (N4-pentyloxycarbonyl-5⬘-deoxy5-fluorocytidine; Xeloda) is an effective and well tolerated
treatment for head and neck (1), colorectal (2– 4), and breast
(4 – 6) cancer. After oral administration, capecitabine passes
rapidly and extensively through the intestinal membrane as an
intact molecule. After absorption, it is first transformed to
5⬘-deoxy-5-fluorocytidine by hepatic carboxylesterase and then
to 5⬘-DFUR3 by cytidine deaminase, which is found in high
concentrations in many human tumor tissues as well as in
healthy liver tissue. As a key step, 5⬘-DFUR is then converted
into 5FU by TP, which is present at higher concentrations in
tumors than in healthy tissues (7). 5FU is catabolized by the
enzyme DPD, which is expressed in liver tissue and tumors (8).
Both experimental (9) and clinical data (10) have shown that
elevated tumor DPD levels confer resistance to 5FU-based
treatment. Experimental data suggest that the cytotoxic efficacy
of capecitabine is improved in cells expressing high levels of TP
and low levels of TS and DPD (11). Clinical data have recently
confirmed this experimental observation by demonstrating the
role of tumor TP and DPD for predicting the efficacy of 5⬘DFUR in the treatment of patients with colorectal cancer (12).
An assessment of tumor up-regulation of TP may help optimize
capecitabine antitumor effects (13–15). Taxanes were found to
up-regulate the tumoral activity of TP and have shown synergistic cytotoxic activity when combined with capecitabine (13,
14). This synergy has recently translated into interesting clinical
results with the docetaxel-capecitabine combination (15).
TP and TS cellular levels are dependent on tumor prolif-
Received 11/4/02; revised 5/5/03; accepted 5/24/03.
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.
Presented in part at the 93rd Annual Meeting of the American Association for Cancer Research, Inc.
1
To whom requests for reprints should be addressed, at Oncopharmacology Unit, Centre Antoine Lacassagne, 33 Avenue de Valombrose,
06189 Nice Cedex 2, France. Phone: 33-4-92-03-15-53; Fax: 33-4-9381-71-31; E-mail: [email protected].
2
Iressa is a trademark of the AstraZeneca group of companies.
3
The abbreviations used are: 5⬘-DFUR, 5⬘-deoxy-5-fluorouridine; CI,
combination index; DPD, dihydropyrimidine dehydrogenase; EGFR,
epidermal growth factor receptor; 5FU, 5-fluorouracil; MTT, 3-(4-5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PSAU, phenylselenenylacyclouridine; TP, thymidine phosphorylase; TS, thymidylate synthase; HPLC, high-performance liquid chromatography;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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4736 ZD1839 in Combination with 5⬘-DFUR
eration and growth (16, 17). Thus, modifying tumor cell proliferation might help optimize the activity of capecitabine through
a modulation of TP and TS. The EGFR cellular pathway regulates cell growth as well as survival (18), and EGFR-targeting
agents are currently in clinical development (19). Because
EGFR is more highly expressed in tumors than normal tissue
(20), tumor selectivity is a possible benefit of EGFR-targeting
agents. The use of EGFR-directed drugs could be an interesting
approach to modulate TS and TP. ZD1839 (Iressa) is a p.o.active and -selective EGFR-tyrosine kinase inhibitor that blocks
signal transduction pathways implicated in proliferation and
survival of cancer cells, and other host-dependent processes
promoting cancer growth. ZD1839 exhibits a broad spectrum of
antitumor activity against many human solid tumor xenografts
including breast, pancreas, lung, colorectal, and head and neck
cancer (21, 22).
The aim of the present study was to examine the effects of
ZD1839 on TS, TP, and DPD in CAL33, a head and neck tumor
cell line that expresses high levels of EGFR (23). In addition,
the cytotoxic effects of combining ZD1839 with 5⬘-DFUR were
examined. This preclinical work was undertaken to assess the
potential for clinical investigations of ZD1839 combined with
the oral fluoropyrimidine capecitabine as a new strategy of
p.o.-administered anticancer treatment.
MATERIALS AND METHODS
Chemicals. ZD1839 and 5⬘-DFUR were kindly provided
by AstraZeneca and Roche Laboratories Inc., respectively. A
50-mM working solution in DMSO was prepared before use.
DMEM and glutamine were purchased from Whittaker (Verviers, Belgium). Fetal bovine serum was obtained from Dutscher (Brumath, France). Penicillin and streptomycin were from
Meyrieux (Lyons, France). Transferrin and insulin were purchased from Flow (Irvine, Scotland). BSA, MTT, and DMSO
were purchased from Sigma (St. Quentin Fallavier, France).
Tritiated 5⬘-DFUR (5.1 Ci/mmol) was purchased from Moravek
Biochemicals (Brea, CA).
Drug Administration Schedule. The human head and
neck cancer cell line model CAL33 was used in the present
study, as in our previous work (23). CAL33 cells carry a p53
mutation (codon 175, CGC 3 CAC) and exhibit high EGFR
expression (34,000 fmol/mg protein). CAL33 has no intrinsic
mitogen-activated protein kinase activity or K-ras mutation
(23).
Cells were routinely cultured in DMEM supplemented with
10% fetal bovine serum, 2 mM glutamine, 50,000 units/liter
penicillin, and 80 ␮M streptomycin in a humidified incubator
(Sanyo, Japan) at 37°C with an atmosphere containing 8% CO2.
The first part of the study consisted of the exploration of
the impact of ZD1839 on the enzyme activities (TS, TP, DPD).
For these experiments, CAL33 cells were seeded in 175-cm2
tissue culture flasks at 1.86 ⫻ 106 cells to obtain exponential
growth for the duration of the experiments. Forty-eight hours
after seeding, CAL33 cells were incubated with ZD1839 at 6 ␮M
(IC50) alone for 96 h. Enzyme activities and gene expression
were analyzed 24, 48, 72, and 96 h after the onset of drug
application. All investigations were tripled in separate experiments for all cellular parameters studied. Attention was paid to
the time- and concentration-related effects of ZD1839 on TP
activity. For these experiments, CAL33 cells were seeded as
described previously. Forty-eight hours after seeding, CAL33
cells were incubated with increasing doses of ZD1839 ranging
from 8 ⫻ 10⫺8 to 2 ⫻ 10⫺6 M and TP activity was analyzed 24,
48, 72, and 96 h after the onset of ZD1839 application.
The second part of the study consisted of the examination
of the sequence-dependent cytotoxic effects resulting from the
combination of ZD1839 and 5⬘-DFUR. Cells were seeded in
96-well microtitration plates (100 ␮l/well) to obtain exponential
growth for the duration of the experiments (initial cell density
was 3000 cells/well). After 48 h, cells were exposed to ZD1839
and/or 5⬘-DFUR in a sequence based on the previously published synergistic interaction observed between ZD1839 and
5FU (24). ZD1839 was given alone for 48 h, then 5⬘-DFUR was
added for 48 h, giving a total length of exposure of 96 h for
ZD1839 and 48 h for 5⬘-DFUR. Eleven concentrations were
tested for each drug: ZD1839 up to 200 ␮M and 5⬘-DFUR from
0.035 to 1000 ␮M. Thereafter, growth inhibition was assessed
48 h after the end of the drug exposure (in medium alone) by the
MTT test (25), as follows. Cells were washed with PBS and
incubated with MTT; after 2 h of exposure, DMSO (100 ␮l) was
added to terminate the reaction, and absorbance at 540 nm was
measured using a microplate reader (Labsystems, Helsinki, Finland). Results were expressed as the relative percentage of
absorbance compared with controls without drug. Cell sensitivity to ZD1839 and 5⬘-DFUR was expressed as IC50 (25). Experimental conditions were tested in sextuplicate (six wells of
the 96-well plate per experimental condition) and in three separate experiments. Dose-effect curves were analyzed using
Prism software (GraphPad Software, San Diego, CA).
CI Calculations. The cytotoxic effects obtained with the
different ZD1839/5⬘-DFUR combinations were analyzed, according to the method of Chou and Talalay (26), on Calcusyn
software (Biosoft, Cambridge, United Kingdom). Interaction
between ZD1839 and 5⬘-DFUR was assessed by means of an
automatically computed CI. The CI was determined at 50% and
75% cell death and was defined as:
CIA⫹B ⫽ [(DA/A ⫹ B)/DA] ⫹ [(DB/A ⫹ B)/DB]
⫹ [␣(DA/A ⫹ B ⫻ DB/A ⫹ B)/DADB],
where CIA⫹B is the CI for a fixed effect (F) for the combination
of cytotoxic A and cytotoxic B; DA/A⫹B is the concentration of
cytotoxic A in the combination A ⫹ B, giving an effect F;
DB/A⫹B is the concentration of cytotoxic B in the combination
A ⫹ B, giving an effect F; DA is the concentration of cytotoxic
A alone, giving an effect F; DB is the concentration of cytotoxic
B alone, giving an effect F; ␣ is the parameter with value 0 when
A and B are mutually exclusive and 1 when A and B are
mutually nonexclusive.
Synergism is indicated by CI ⬍ 0.8, additivity by CI values
between 0.8 and 1.2, and antagonism by CI ⬎1.2; slight synergistic and additive cytotoxic activity are indicated by CI values
of 0.8 and 1.2, respectively.
For the application of the Chou and Talalay model (26), it
is recommended that the cytotoxic agents are used at a fixed
dose ratio (for example, ratio of drug IC50).
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Clinical Cancer Research 4737
Cell Cycle Analysis. The cell cycle was analyzed by
fluorescence-activated cell sorting according to the Vindelov
model. For flow cytometry analysis, 106 CAL33 cells in exponential growth were treated with graded concentrations of
ZD1839 for 24 h and then washed three times with citrate
buffer. Cell pellets were incubated with 250 ␮l of trypsincontaining citrate buffer for 10 min at room temperature and
then incubated with 200 ␮l of citrate buffer containing a trypsin
inhibitor and RNase (10 min) before adding propidium iodide
(200 ␮l at 125 ␮g/ml). Samples were analyzed on a Becton
Dickinson FACScan flow cytometer using Modfit software,
which was also used to determine the percentage of cells in the
different phases of the cell cycle. Propidium iodide was excited
at 488 nm, and fluorescence was analyzed at 620 nm (fluorescence channel 3).
TS Activity Assay. TS activity was measured according
to the tritium-release assay described by Spears and Gustavsson
(27). Cytosol (25 ␮l) was incubated with [3H] 2⬘deoxyuridine5⬘-monophosphate (final concentration, 1 ␮M) and 5,10-methylenetetrahydrofolate (final concentration, 0.62 mM) in a total
volume of 55 ␮l. After 0 (for blank subtraction), 10, 20, and 30
min of incubation at 37°C, the reaction was stopped in ice.
Excess [3H]2⬘deoxyuridine-5⬘-monophosphate was removed by
adding activated charcoal (300 ␮l, 15%) containing 4% trichloroacetic acid before a 5-min centrifugation at 14,000 ⫻ g at
room temperature. The [3H]2O formed during the incubation
was then counted in the supernatant by a liquid scintillation
counter (Wallac 1409 DSA; Wallac, Turku, Finland). Results
were expressed as femtomoles of [3H]2O formed per minute per
milligram of protein, based on the linear regression obtained
from the incubation times. The sensitivity limit was 10 fmol/
min/mg protein. Interassay reproducibility was evaluated
through repeated analysis of single-use aliquots of a pooled
cytosol: n ⫽ 5; mean, 1110 fmol/min/mg protein; SD, 78.59
fmol/min/mg protein; coefficient of variation, 7.08%.
TP Activity Assay. There are two distinct pyrimidine
nucleoside phosphorylases present in normal and neoplastic
cells: TP, for which the major substrate is thymidine, and
uridine phosphorylase, which is responsible for the reversible
catalysis of uridine to uracil. Because TP is mainly responsible
for the catalysis of 5⬘-DFUR into 5FU, TP activity was measured in the analyzed samples. Specific inhibitors for TP (TP
inhibitor) and uridine phosphorylase (PSAU) were applied to
determine the specific activity of TP in the reaction mixture.
PSAU was kindly provided by Dr. M. El Kouni (University of
Alabama at Birmingham, Birmingham, AL).
The analytical method used for the determination of TP
activity was derived from Kubota et al. (16). Cultured cells
(107) were homogenized in 500 ␮l of lysis buffer [50 mM
Tris-HCl (pH 6.8), 1% Triton X-100, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.02% mercaptoethanol]. The
samples were centrifuged at 105,000 ⫻ g for 30 min at 4°C.
Protein concentration was determined by using the method of
Bradford. Supernatants (0.8 mg of protein/ml) were incubated
for 4 h at 37°C with 10 mM 5⬘-DFUR and 180 mM potassium
phosphate (pH 7.4) ⫾ 100 ␮M TP inhibitor or PSAU. The
reaction was stopped by the addition of 360 ␮l of ice-cold
methanol to the 120-␮l reaction mixture. After removal of the
precipitate by centrifugation, an aliquot (dilution, 1:5) of the
reaction mixture was applied to the HPLC column (Licrospher
100-RP 18). The elution buffer consisted of 50 mM phosphate
buffer (pH 6.8) containing 10% methanol. The amount of 5FU
produced was monitored by UV absorbance (262 nm). TP
activity was expressed as nanomoles of 5FU converted/milligram of protein per hour.
DPD Activity Assay. DPD activity was measured according to the method described by Harris et al. (28) and under
experimental conditions previously described by us (29).
DPD activity determination consisted of the quantification of
14
C-dihydro-5-fluorouracil, 14C-fluoro-␤-alanine, and 14C-␣fluoroureidopropionic acid using a previously reported HPLC
method (29). DPD activity was calculated by summing the
dihydrofluorouracil, fluoro-␤-alanine, and ␣-fluoroureidopropionic acid peaks. DPD activity was expressed as picomoles of
(14C 5-fluorouracil) catabolized per minute and per milligram of
protein. Each sample was assayed in duplicate, and DPD activity was measured in three independent experiments. The sensitivity limit was 1 pmol/min/mg protein. The interassay reproducibility (pooled cell suspension) gave a coefficient of
variation of 12% (n ⫽ 8).
TS, TP, and DPD Expression. mRNA levels of TS, TP,
and DPD were measured by real-time quantitative RT-PCR on
the samples obtained at 24, 48, 72, and 96 h after the onset of
ZD1839 application on CAL33 cells.
RNA Extraction and Reverse Transcription. DPD,
TS, and TP expressions were determined in cell line pellets
stored at ⫺80°C. Total RNA was isolated from 4 ⫻ 106 cells
using the RNA NOW kit from Biogentex (Ozyme, MontignyleBretonneux, France) based on a method derived from Chomczynski and Sacchi (30). RNA quality was checked by agarose
gel electrophoresis. Quantification was performed by densitometric analysis at 260 nm. Total RNA (100 ng) was incubated
for 10 min at 25°C in a 20-␮l final volume of 50 mM Tris-HCl
(pH 8.3), 75 mM KCl, 3 mM MgCl2, containing 0.4 mM of each
deoxyribonucleotide triphosphate, 0.08 A260 units of random
hexamers, 20 units of avian myeloblastosis virus reverse transcriptase, and 40 units of RNase inhibitor. This preparation was
then incubated for 1 h at 42°C, followed by 5 min at 94°C in a
classical Biometra thermocycler (all of the reagents for the RT
reaction were included in the mRNA amplification kit from
Roche Diagnostics, Meylan, France).
Primers. The oligonucleotides used for DPD, TP, and TS
amplification are the property of Roche. Primers for DPD, TP,
and TS gave products of 148 bp, 108 bp, and 111 bp, respectively.
The GAPDH primers (GADPH is the reference gene in all
three kits) gave a product of 123 bp.
PCR Conditions. LightCycler mRNA Quantification
kits (Roche Molecular Biochemicals) were used to perform
real-time RT-PCR for DPD, TP, and TS by using the LightCycler system. The manufactured kit contained 25 mM MgCl2,
LightCycler DNA Master SYBR Green I 10X containing deoxynucleotide triphosphate mix, 10 mM MgCl2, SYBR Green I
dye, specific primers for the target and the reference gene, and
Hot Start TaqDNA polymerase. cDNA (4 ␮l), previously obtained by RT-PCR (see above), was amplified in duplicate (one
incubation for the target gene, one incubation for the reference
gene) in a 20-␮l reaction capillary containing 2 ␮l of Light-
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4738 ZD1839 in Combination with 5⬘-DFUR
Table 1
Time-related influence of ZD1839 (6 ␮M) on proliferation
of CAL33 cells
Time (h)
% G0–G1,
mean (SD)a
% S, mean
(SD)a
% G2-M,
mean (SD)a
24
48
72
96
59 (11)
69 (6)
77 (4)
87 (3)
35 (11)
22 (2)
15 (5)
9 (3)
7 (1)
9 (5)
7 (2)
4 (1)
24
48
72
96
89 (10)
86 (6)
87 (2)
87 (12)
7 (3)
8 (2)
7 (0)
7 (9)
4 (1)
6 (2)
6 (1)
7 (2)
Control
ZD1839
a
Results from three separate experiments.
Cycler DNA Master SYBR Green I 10X, 1.6 ␮l of 25 mM
MgCl2, and 6 ␮l of one couple of primers. In each experiment,
a cDNA preparation obtained from a RNA sample, included in
the kit, was introduced as the calibrator. Simultaneously, a blank
was performed by incubating H2O PCR grade instead of sample
cDNA.
The PCR protocol programmed on the LightCycler software consisted of two steps. The first step was denaturation of
cDNA and activation of the enzyme for 5 min at 95°C. In the
second step, DNA was amplified for 40 cycles of 10 s at 95°C,
10 s at 62°C, and 10 s at 72°C. Results, calculated using
RelQuant software, were expressed as the ratio of concentration
of TP (or DPD or TS) to GAPDH for a given sample, normalized by the same ratio for the calibrator.
Determination of [3H]-5ⴕ-DFUR Intracellular Metabolites. Separation and detection of [3H]]-5⬘-DFUR metabolites
was performed as described previously (31). Exponentially
growing cells were exposed for 24 h to either tritiated 5⬘-DFUR
alone or after a 48-h pretreatment period with ZD1839. Drug
concentrations were set at their respective IC50. Cells were
harvested after the 24-h exposure to 5⬘DFUR, and cytosols were
isolated for HPLC analysis. The HPLC consisted of a HP1090
(Hewlett Packard) system coupled with a A200 radioactive flow
detector (Packard). Separation of tritiated metabolites was obtained using a Lichrospher 100 RP18 5-␮M column (Hewlett
Packard) eluted by 50 mM K2HPO4 (pH 6.8) containing 5 mM
tetrabutyl ammonium nitrate and 5% (0 –32 min) to 25% (32– 60
min) methanol. Results were expressed as dpm/␮g protein.
Statistical Analysis. Differences between mean values
were evaluated using either one-way ANOVA with Tukey test
or one-way ANOVA on ranks with Dunnett’s test or StudentNewman-Keuls test, according to data distribution. Correlation
coefficients (r) from linear regressions and respective Ps (P ⬍
0.05 was regarded as statistically significant) were computed
using the program SPSS (Paris, France).
RESULTS
The impact of ZD1839 on cell cycle organization is shown
in Table 1. Compared with drug-free controls, ZD1839 increased the proportion of cells in G0-G1 and decreased the
proportion in S phase. ZD1839 slowed down cell proliferation
after drug exposure and caused a rapid increase in G0-G1 phase
Fig. 1 ZD1839 induces an early and marked reduction in TS activity.
TS activity was markedly inhibited (40-fold) as early as 24 h after
ZD1839 exposure, and this inhibition was maintained for 72 h. ⴱ, P ⬍
0.05 versus control. Bars, SD from mean of triplicates.
Fig. 2 ZD1839 (6 ␮M) induces a time-related increase in TP activity
with the maximum effect 48 h after drug onset. ⴱ, P ⬍ 0.01 versus
control. Bars, SD from mean of triplicates.
(with a maximum of 89% cells in G0-G1 phase at 24 h). This
phenomenon was accompanied by a decrease in S phase (with a
minimum of 7% of cells in S phase at 24 h) compared with
control. Cells treated with ZD1839 presented a statistically
significant maximum decrease of 40-fold in TS activity at 24 h
(P ⫽ 0.0001; Fig. 1) and a statistically significant maximum
increase of almost 2-fold in TP at 48 h (P ⫽ 0.01; Fig. 2).
ZD1839 treatment did not significantly modify DPD activity at
any time of exposure (the ratio DPD activity in ZD1839-treated
cells/control cells remained close to 1 from 24 h to 96 h after
ZD1839 onset; data not shown). A more detailed examination of
the effects of ZD1839 on TP activity revealed time- and concentration-related changes in TP in the presence of ZD1839
(Fig. 3). Although TP activity was significantly up-regulated as
a function of both ZD1839 concentration and time of exposure
to ZD1839 (P ⫽ 0.0001), it is clear that marked changes relative
to controls occur above the micromolar level in ZD1839. In this
situation, the maximal relative increase in TP at 48 h is again put
into evidence.
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Clinical Cancer Research 4739
Fig. 3 There were statistically significant time- and concentrationrelated increases in TP activity under the presence of ZD1839. P ⫽
0.0001, test of rank for both time-dependent and ZD1839 concentrationdependent effects. Bars, SD from mean of triplicates
Fig. 4 mRNA levels of TP were modified in parallel with the enzyme
activity (r2 ⫽ 0.98; P ⫽ 0.0068); bars, SD from mean of triplicates.
Experimental points were 24, 48, 72, and 96 h after ZD1839 exposure
(see “Materials and Methods”).
To elucidate the origin of the modifications in enzyme
activities observed, the respective gene expressions were examined in parallel. A significant link between TP activity and TP
expression levels was demonstrated with an r2 value of 0.98
(P ⫽ 0.0068; Fig. 4). Modifications in TS activity and TS
expression levels after ZD1839 treatment were also positively
correlated (r2 ⫽ 0.68), but the correlation was not significant
(data not shown).
Monitoring of 5⬘-DFUR metabolism showed a more efficient activation of this fluoropyrimidine, both quantitatively and
qualitatively, when this later drug was combined with ZD1839
(Fig. 5). In particular, cells exposed to ZD1839 ⫹ 5⬘-DFUR
displayed higher amounts of 5FU derivatives, FUR and fluorodeoxyuridine, as compared with those produced after 5⬘-DFUR
alone. As a corrolary, nuclear incorporation of triphosphorylated
fluoronucleotides was found to be increased by up to 110%
when cells were exposed to the combination 5⬘-DFUR ⫹
ZD1839 in comparison with 5⬘-DFUR alone (data not shown).
Dose-effect curves resulting from the application of
ZD1839 alone, 5⬘-DFUR alone, or both drugs on the CAL33
cell line are shown in Fig. 6. Both drugs exhibited classic
dose-response curves with IC50 for ZD1839 and 5⬘-DFUR of
2.38 ␮M and 4.8 ␮M, respectively. CIs (mean ⫾ SD) from
5⬘-DFUR-ZD1839 combinations were 0.58 ⫾ 0.1 and 0.63 ⫾
0.1 for 50% survival (CI50) and 25% survival (CI75), respectively. An illustration of the synergistic cytotoxic effects between ZD1839 and 5⬘-DFUR is shown in Fig. 7. Altogether,
these data point to a strong synergistic interaction between
ZD1839 and 5⬘-DFUR when ZD1839 is applied before and
during 5⬘-DFUR application.
concentrations 4-fold higher than in control tumors (32). In
WiDr human colon cancer xenografts, vinblastine, vindesine,
and cisplatin were found to up-regulate TP activity (14), and
gemcitabine and vinorelbine have also been demonstrated to
cause TP up-regulation (14). Similarly, TP concentrations were
increased up to 10-fold after exposure to human recombinant
IFN-␥ (14). Radiotherapy has also been shown to enhance TP
expression in tumor tissue in a number of human cancer xenografts, with single-dose irradiation resulting in a 13-fold increase in intratumoral TP activity, whereas no up-regulation was
observed in healthy liver tissue (33). However, it is not clear
from these studies what the origin of the up-regulation in TP
activity is; most of the effects on TP were observed in xenograft
models, whereas up-regulation of TP by these drugs was hardly
detectable in cultures of tumoral cells (13, 33). The hypothesis
was that elevations in TP observed in vivo and not in vitro could
be mediated by tumor necrosis factor ␣ in tumor cells, which in
turn may up-regulate TP (13). The present study clearly establishes that TP activity may be intrinsically up-regulated in the
tumor cells by a change in proliferation status induced by
ZD1839, a drug that specifically targets EGFR. The role of
ZD1839 in TP up-regulation was further demonstrated by the
clear time- and concentration-dependent effects of ZD1839 on
TP activity (Fig. 3). Parallel to TP were the modifications
observed in TS activity, but in an opposite sense: a decrease in
TS activity after a ZD1839-induced reduction in cell proliferation. This decrease in TS activity induced by the drug-related
inhibition in cell proliferation agrees well with previously reported data showing the link between tumor cell proliferation
and TS activity (34 –36). In the present study, a positive and
significant relationship was put into evidence between TS activity and the percentage of cells in S phase (r2 ⫽ 0.87; P ⫽
0.0008; n ⫽ 7; data not shown). The fact that parallel changes
occurred in enzyme activities and enzyme expressions (mRNA
levels) means that the observed modifications are mainly under
transcriptional control. It has been postulated that TS might
regulate the transcription of cellular gene expression including
p53 and perhaps other cell cycle-related proteins (37). It is, thus,
DISCUSSION
In a study of mouse xenograft models of human breast and
colon cancers, exposure to paclitaxel, docetaxel, or mitomycin C
increased tumor concentrations of TP by 4- to 8-fold within 8
days of administration (13). Another study showed that daily
oral administration of cyclophosphamide for 2 weeks resulted in
a gradual increase in TP concentrations in tumor tissue, reaching
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Research.
4740 ZD1839 in Combination with 5⬘-DFUR
Fig. 5 Formation of intracellular 5⬘-DFUR derivatives. Cells were exposed during 24 h to tritiated 5⬘-DFUR alone (䡺) or after a 48-h pretreatment with ZD1839 (u). Drug concentrations were
set at their respective IC50. Experiments were performed as described in “Materials and Methods.”
Values are from three experiments performed at a
distance (vertical bars, SD). FU-H2, dihydrofluorouracil; FUdR, fluorodeoxyuridine; FdUMP,
fluorodeoxyuridine monophosphate; FUTP, fluorouridine triphosphate; FdUTP, fluorodeoxyuridine triphosphate.
Fig. 6 Survival curves for CAL33 cells show a marked shift to the left
for the curve representing the association ZD1839 plus 5⬘-DFUR. Dose
ranges were 7 ⫻ 10⫺8 M to 7 ⫻ 10⫺4 M for ZD1839 and 3.5 ⫻ 10⫺7 M
to 3.5 ⫻ 10⫺3 M for 5⬘-DFUR, and the drug concentration ratio in the
combination was that of their IC50 ratio (IC50 5⬘DFUR ⫽ 5 ⫻ IC50
ZD1839). CIs [Chou and Talalay model (26)] were 0.58 ⫾ 0.1 and
0.63 ⫾ 0.1 at 50% and 25% cell survival, respectively.
possible that changes in TS may participate in the final modification in TP activity with a possible translational repression of
TP under the presence of TS; a decrease in TS protein would
allow more TP mRNA to be translated into TP protein. A
possible TS protein-TP mRNA interaction would be interesting
to explore in additional experiments, which were beyond the
scope of the current study. The next logical step in the present
study was to analyze the cytotoxic effect resulting from the
combination of ZD1839 with 5⬘-DFUR. A strong synergistic
interaction in terms of final cytotoxic effect was observed when
combining these two drugs (Fig. 6); this is in line with both a
reduction in TS activity by ZD1839, which is in favor of
fluoropyrimidine activity (38), and an increase in TP activity,
which permits more 5FU to be intracellularly delivered from its
prodrug 5⬘-DFUR (7). This last hypothesis was strongly supported by the monitoring of the intracellular metabolism of
5⬘-DFUR. Marked differences in cellular 5FU anabolites were
observed in favor of the combination ZD1839 –5⬘DFUR as
compared with 5⬘-DFUR alone (Fig. 5). Above all, these obser-
Fig. 7 Isobolographic analysis on the CAL33 cell line for the association ZD1839, followed 48 h later by 5⬘-DFUR. The space between
curves represents the zone of additivity (30% cell survival). The symbol
(⫹) results from an experimental condition with ZD1839 at 2 ⫻ 10⫺6 M
and 5⬘-DFUR at 1 ⫻ 10⫺5 M. The symbol (⫹) is located in the area of
synergistic interaction.
vations are fully consistent with previously published reports
showing that higher levels of TP in the target cell are related to
an increase in the cytotoxic effect of capecitabine, 5⬘-DFUR,
and 5FU (39 – 41). The present data may serve as a rational basis
for setting up clinical trials combining EGFR-targeting agents
such as ZD1839 with the new oral fluoropyrimidine capecitabine, which is the prodrug of 5⬘-DFUR. Such strategy of drug
combination would carry a double advantage: the theoretical
advantage of tumor selectivity, because high EGFR expression/
signaling plays a more important role in tumor tissue than
normal tissue (20), and the oral administration route of capecitabine plus ZD1839 therapy.
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ZD1839 (Iressa) Modifies the Activity of Key Enzymes Linked
to Fluoropyrimidine Activity: Rational Basis for a New
Combination Therapy with Capecitabine
Nicolas Magné, Jean-Louis Fischel, Alain Dubreuil, et al.
Clin Cancer Res 2003;9:4735-4742.
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