synergistic interaction between cisplatin and gemcitabine in vitro1 · addp, 12,000 cells/well;...
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Vol. 2, 521-530, March 1996 Clinical Cancer Research 521
Synergistic Interaction between Cisplatin and Gemcitabine in Vitro1
Andries M. Bergman,
Veronique W. T. Ruiz van Haperen,
Gijsbert Veerman, Catharina M. Kuiper,
and Godefridus J. Peters2
Department of Oncology, Free University Hospital, P.O. Box 7057,
1007 MB Amsterdam, the Netherlands
ABSTRACT
2’,2’-Difluorodeoxycytidine (dFdC; gemcitabine) is a
new antineoplastic agent that is active against ovarian car-
cinoma, non-small-cell lung carcinoma, and head and neck
squamous cell carcinoma. cis-diamminedichioropiatinum
(CDDP; cisplatin) is used commonly for the treatment ofthese tumors. Because the two drugs have mechanisms of
action that might be complementary, we investigated a pos-
sible synergism between dFdC and CDDP on growth inhi-
bition. The combination was tested in the human ovarian
carcinoma cell line A2780, its CDDP-resistant variant ADDP
and its dFdC-resistant variant AG6000, the human head
and neck squamous cell carcinoma cell line UMSCC-22B,and the murine colon carcinoma cell line C26-10. The cellswere exposed to dFdC and CDDP as single agents and to
combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h
with a total culture time of 72 h. Synergy was evaluated
using the multiple drug effect analysis. In A2780 and ADDP
cells, simultaneous exposure to the drugs for 24 and 72 h
resulted in synergism, but shorter exposure times were an-
tagonistic. No synergism was found in the UMSCC-22B andC26-10 cell lines at prolonged simultaneous exposure. How-
ever, a preincubation with CDDP for 4 h followed by a dFdC
incubation for 1, 4, 24, and 72 h was synergistic in all cell
lines except C26-10 cells. A 4-h preincubation with dFdC
followed by an incubation with the combination for 20 and68 h was synergistic in all cell lines. Initial studies of themechanism of interaction concentrated on the effect of
CDDP on dFdCTP accumulation and DNA strand breakformation. In all cell lines, CDDP failed to increase dFdCTP
accumulation at 4- or 24-h exposure to dFdC; in two cell
lines, CDDP even tended to decrease dFdCTP accumulation.
Neither dFdC nor CDDP caused more than 25% double
strand break formation, whereas in the combination, CDDP
even tended to decrease this type of DNA damage. The
synergistic interaction between the two drugs is possibly the
result of dFdC incorporation into DNA and/or CDDP-DNA
adduct formation, which may be affected by each other.
INTRODUCTION
dFdC3 is a deoxycytidine analogue with two fluorine atoms
substituted for the two hydrogens at the 2’ position of the ribose
ring. The compound has established activity against solid tu-
mors, as described for several solid tumor models, including
ovarian and head and neck cancer (1-3). In the clinical setting,
dFdC has proven to be effective against ovarian cancer and
non-small cell lung cancer and moderately effective against
head and neck cancers (4-6). After entering the cell, the drug
has to be phosphorylated by dCK to dFdCMP and subsequently
to dFdCTP (7), which can be incorporated into both DNA and
RNA (8, 9). After incorporation of dFdCTP, one more de-
oxynucleotide can be incorporated, after which DNA polymer-
ization stops (9). Unlike ara-C, exonuclease activity is unable to
excise dFdCMP from the DNA (9). dFdC can be inactivated by
the action of deoxycytidine deaminase to 2’,2’-difluorode-
oxyuridine (7).
For the treatment of the above-mentioned tumors, CDDP, a
square planar coordination compound, is an important agent
(10). The antitumor activity of CDDP is the result of the
formation of adducts within the DNA (1 1-17). The binding of
the CDDP moiety with two adjacent guanines on the same DNA
strand is the most abundant lesion and is held responsible for the
antitumor activity (12, 13). This lesion is thought to introduce a
distortion in the DNA that is large enough to stop the division of
the cells without being recognized rapidly and, thus, removed
efficiently by repair enzymes (18). There is a possible relation
between CDDP-DNA adduct levels and cell growth inhibition in
cultured cells (14, 15).
Synergistic interactions of CDDP with ara-C and DAC,
two other deoxycytidine analogues, have been described previ-
ously. Studies on the mechanism of the synergism suggested
differences in the interactions between ara-C and CDDP and
DAC and CDDP (19, 20). CDDP-DNA adduct formation was
enhanced by DAC incorporation, but ara-C did not affect the
binding of CDDP to DNA. However, ara-C delayed the recov-
ery of DNA synthesis inhibited by CDDP markedly. Because
dFdC is related structurally to ara-C and DAC, it seemed ap-
propriate to investigate the combination of CDDP with this
compound too. One possible mechanism for synergy could be
the inhibitory effect of CDDP on ribonucleotide reductase (21),
possibly causing a depletion of dCTP pools, a potent feedback
inhibitor of dCK. Subsequently, this could lead to increased
dFdC phosphorylation. Alternatively, DNA might become more
accessible to DNA adduct formation by distortions caused by
Received 6/26/95; revised 1 1/15/95; accepted 12/5/95.I This work was supported by grants from the Dutch Cancer Society by
Grants IKA-VU 90-19 and VU 94-753, and by a grant from Eli Lilly,
Inc. (Nieuwegein, the Netherlands).2To whom requests for reprints should be addressed. Phone: + 31-20-
4442633; Fax: +31-20-4443844.
3 The abbreviations used are: dFdC, 2’,2’-difluorodeoxycytidine (gem-
citabine); dCK, deoxycytidine kinase; ara-C, i-�3-D-arabino furanosylcytosine; CDDP, cis-diamminedichloroplatinum (cisplatin); DAC, 2’-de-oxy-5-azacytidine; 22B, UMSCC-22B; TCA, trichloroacetic acid; SRB,
sulphorhodamine B; IC50, 50% inhibitory concentration; CI, combination
index; FA, fraction affected; Dm, dose required to produce a 50% growthinhibition; FADU, fluorometric analysis of DNA unwinding.
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522 Synergistic Interaction between Cisplatin and Gemcitabine
dFdCTP incorporation. Furthermore, because both drugs have
different, nonoveriapping side effects, synergistic or even addi-
tive antitumor effects of the combination may be clinically
worthwhile. In addition, when using combinations, it is less
likely that resistance will develop.
Here, we describe the in vitro synergism between dFdC and
CDDP. Three human ovarian carcinoma cell lines, the dFdC-
and CDDP-sensitive A2780 cell line and its CDDP- and dFdC-
resistant variants ADDP and AG6000, respectively, the human
head and neck squamous cell carcinoma cell line 22B, and the
dFdC-insensitive murine colon tumor cell line C26-lO were
exposed to both drugs in different concentrations and schedules.
To investigate possible mechanisms of synergism, several pa-
rameters known to be important in the action of dFdC were
studied, and the effect of CDDP on the dFdCTP accumulation
and the number of DNA strand breaks induced by dFdC were
measured to clarify the mechanism of the synergistic interaction.
MATERIALS AND METHODS
Chemicals and Reagents. DMEM and RPMI 1640 were
purchased from Flow Laboratories (Irvine, United Kingdom);
FCS was from GIBCO (Grand Island NY), TCA, glutamine, and
gentamicin were from Merck (Darmstadt, Germany), trypsin,
and SRB was from Sigma Chemical Co. (St. Louis, MO). dFdC
was kindly supplied by Eli Lilly Research Labs (Indianapolis,
USA). CDDP was purchased from Bristol-Myers Squibb
(Weesp, the Netherlands). All other chemicals were of analyti-
cal grade and commercially available.
Cell Culture. The in vitro experiments were performed
with five different cell lines: A2780, a human ovarian cancer
cell line (22, 23), and two variants, ADDP, with a 50-fold
induced resistance to CDDP (kindly provided by Dr. K. J.
Scanlon City of Hope National Medical Center, Duarte, CA;
Ref. 22), and AG6000, with a 150,000-fold induced resistance
to dFdC (23); 22B, a human head and neck squamous cell
carcinoma cell line (24); and the murine colon carcinoma cell
line C26-lO (25). Doubling times of the cell lines were 21, 32,
37, 40, and 14 h, respectively. Cells were grown in monolayers
in DMEM supplemented with 5% heat-inactivated FCS, 1 mM
L-glutamine, and 250 ng/ml gentamicin, except for ADDP,
which was cultured in RPMI 1640 medium supplemented with
10% heat-inactivated FCS, 1 mM L-giutamine, and 250 ng/ml
gentamicin at 37#{176}Cat 5% CO2.
Chemosensitivity Testing. The determination of the
IC50 was performed using the SRB assay (26, 27). The assay
was performed using the National Cancer Institute protocol with
some small modifications (27). Culture conditions were opti-
mized for each cell line. The five cell lines were exposed to
dFdC and CDDP as separate agents and to a combination of
both. At day 1 , the cells were plated in 96-well plates in
different densities, depending on their doubling times. The
optimal plating number was the highest number of cells possible
to enable log linear growth for 72 h (A2780, 5,000 cells/well;
ADDP, 12,000 cells/well; AG6000, 18,000 cells/well; C26-lO,
1,500 cells/well; and 22B, 15,000 cells/well) in a volume of 100
�i.l/well. Of the 96-well plates, the upper and lower rows (rows
A and H) and the last two columns at the right side (columns 1 1
and I 2) were not used for cells, because we observed evapora-
tion in these wells earlier. Thus, the first column contained only
medium, the second column contained cells not exposed to
drugs, and eight columns contained cells exposed to increasing
concentrations of drugs. For each drug or drug combination,
three wells were used.
On day 2, dFdC and CDDP were added in a volume of 100
pal, resulting in series of final concentrations of 2.10 ‘ I_1.106
M dFdC and l.10_8_5,10_4 M of CDDP. The concentrations in
the combination had the same range as the separate agents, with
a dFdC:CDDP ratio of 1:500, which was based on the separate
IC50 values in the various cell lines. Similar to the National
Cancer Institute protocol, we chose the same culture time for all
cell lines. The cells were exposed for 1 , 4, 24, and 72 h. After
the exposure, the medium in the control and the drug-containing
wells was removed and replaced by fresh, drug-free medium
after a washing step with 50 �il PBS (pH 7.4), and the cells were
cultured until 72 h after the initial drug addition. Care was taken
to ensure that cell loss was minimal in the procedure; further-
more, control wells were also washed. Final A values in washed
and nonwashed wells were hardly different. At the end of the
culture, the cells were precipitated with 50 pi ice-cold 50%
(w/v) TCA and fixed for 60 mm, after which the SRB assay was
performed as described (27). Only proteins of intact cells are
stained, because proteins from killed cells would be degraded to
amino acids by endogenous proteases after lysis (27). Growth
inhibition curves were made relative to control As of every SRB
assay. Two control values were always included, that of the A of
cells at the day of drug administration (day 0, set at 0%) and that
of cells after 3 days (set at 100%). This enabled us to calculate
IC50 values (relative A at 50%), total growth inhibition (A of
drug-treated cells similar to the initial value, 0%) and cell killing
(A of drug treated cells lower than the initial value 0%). The
points were connected by straight lines, and the IC50 values
were determined from the interpolated graph (28). Also, the
method of Chou and Talalay (29) was used; for these cell lines
similar values (Dm) were achieved. The cells were not only
exposed to the combination simultaneously, but two sequential
schedules were also used: (a) 4-h preincubation with CDDP,
after which the cells were exposed to dFdC for 1, 4, 24, or 72 h;
and (b) 4-h preincubation with dFdC, followed by an incubation
with both dFdC and CDDP for 20 or 68 h (Fig. 1).
Dose-response interactions (antagonism, additivity, and
synergism) between dFdC and CDDP were expressed as a
nonexclusive case CI for every FA, using the method of Chou
and Talalay (29), processed by a computer program developed
by Chou and Chou (Biosoft, Cambridge, United Kingdom). This
program was chosen because it provided one of the few objec-
tive computerized evaluation procedures (30). The data are
supported by classic isobolograms made by the same computer
program, which were based on the original method of Steel and
Peckham (3 1 ). Other observations in favor for the choice of this
program were the sigmoidal forms of the growth curves, ideal
for median effect analysis. Thus, IC50 values (by interpolation)
and Dm values (by the computer program) were usually similar.
Measurable IC50 values for each drug separately are not re-
quired for computerized evaluation; Dm values would be calcu-
lated then by the program by extrapolation. For the separate
drugs, we had to introduce their respective growth inhibition
parameters, expressed as FA (e.g. , a FA of 0.25 is a growth
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aim.
dFdC+
CDDP
coop
->dFdc
dFdC.>coop
-4 14 24 72
Clinical Cancer Research 523
I I
I I
‘Sf1 I
I I
�-
time (hr)
Fig. 1 Schematic representation of the drug exposure schedules of thegrowth inhibition experiments. sim. dFdC+CDDP, simultaneous expo-
sure to the combination of dFdC and CDDP; CDDP-*dFdC, preincu-
bation with CDDP followed by dFdC exposure; dFdC-*CDDP, prein-cubation with dFdC alone followed by exposure to the combination of
dFdC and CDDP. D, CDDP; �, dFdC; R, dFdC and CDDP; D. drug
free.
inhibition of 25%). The program was unable to evaluate values
of FA > 1, indicating cell killing or FA = 0, indicating no
growth inhibition. Growth inhibition of 0% of each drug sepa-
rately had to be introduced as a FA of 0.01, whereas total growth
inhibition of 100% had a FA of 0.99. For the final evaluation,
we excluded values of the combination for almost complete
growth inhibition (FA > 0.90) and hardly any effect (FA <
0. 10). The CI was calculated by the formula:
CI = [(D)l/(D5)lJ + [(D)2/(D5)2] + [a(D)l,2/(Dx)i,2]
Where a = 1 for mutually nonexclusive drugs; (D)l, (D)2, and
(D)l,2 are the doses of the separate drugs and their combination
in a fixed ratio of 1:500; and (D�)l, (D�)2 and (D�)l,2 are the
doses resulting in a growth inhibition of x%. These doses are
calculated by the formula: D = D, m X [(FA/l - FA)]/m,
where D� is the dose required to produce a 50% growth inhi-
bition, FA is the fraction affected, and m is the slope of the
median effect plot (a measure of sigmoidicity). A CI < I mdi-
cates synergism, > I indicates antagonism, and a CI of 1 mdi-
cates additivity. Results were also analyzed by isobologram
analysis using the same program as well by simple calculations
as comparisons of expected and observed growth inhibition or
cell killing. All three methods led to comparable conclusions,
underlining the significance of the data obtained with the mul-
tidrug effect analysis.
dFdCTP Accumulation. At day 1 , the cells were plated
in six-well plates in different densities, depending on their
doubling times (A2780, I .5 X 10� cells/well; ADDP, 1 .5 X I 0�
cells/well; AG6000, 2.5 X l0� cells/well; C26-l0, I X l0�
cells/well; and 22B, 1.0 X 106 cells/well) in a volume of 2
mi/well. At day 2, the cells were exposed to either 0. 1 p.M dFdC
or I �J.M dFdC as single agents or to combinations of 0. 1 pM
75’- I
dFdC with 20 �LM CDDP or to 1 J.LM dFdC with 100 p.M CDDP
and incubated at 37#{176}C.After 4 and 24 h of incubation, the cells
were harvested by trypsinization, washed, and counted while
kept on ice. Thereafter, the nucleotides were extracted using
TCA precipitation and alamine and freon neutralization (32).
Finally, the nucleotides were analyzed on high-performance
liquid chromatography using a Partisphere SAX (Whatman,
Clifton, NJ) column with a linear gradient between S m�i
NH4H2PO4 (pH 2.8; buffer A) and 0.5 M NH4H2PO4/0.25 M
KCL (pH 3.0; buffer B; 35-100% B over 30 mm) at a flow rate
of 1 .5 mi/mm. Samples were detected at 254 and 280 nm (32).
FADU Assay. To measure the dFdC-induced DNA dam-
age the FADU assay was used (33, 34). DNA damage was
assessed by exposing lysed cells to an alkaline environment,
allowing the DNA to unwind. The extent of strand breaks in the
DNA determines the extent of DNA unwinding at the end of
incubation. For this purpose, 3-5 X 106 A2780 or 22B cells
were incubated for 24 h at 37#{176}Cwith either I .5 nM dFdC as a
single agent or the combination of 1 .5 nM dFdC and 0.75 fLM
CDDP. For C26-lO cells, a higher drug concentration was
chosen, because for the concentrations used for the A2780 and
22B cell lines, no DNA damage could be detected. Three to S X
106 C26-l0 cells were incubated for 24 h with either 20 nM
dFdC as a single agent or the combination of 1 .5 nM dFdC and
0.75 �iM CDDP. Cells exposed to 50 p.M VP-16 (Etoposide) for
1 h were used as a positive control; cells not exposed to drugs
were used as a negative control.
After harvesting, using trypsmnization, cells were counted
and suspended in 0.25 M meso-inositol, 10 mr�i sodium phos-
phate, and I M MgCl, (pH 7.2) buffer (solution B; 1 .5-2.5 X 106
cells/mi). Every sample was distributed to three tubes (in dupli-
date, 0.2 mi/tube), in which the cells were lysed by 0.2 ml
solution of 9 M urea, 10 mrvi NaOH, 2.5 rn�i cyclohexanediami-
netetraacetate, and 0. 1% SDS (solution C). The cells were
exposed to an alkaline environment by adding 0. 1 ml solution D
(45% solution C and 55% 0.2 M NaOH) and 0.1 ml solution E
(40% solution C and 60% 0.2 M NaOH) gently to the tubes
without mixing and then kept on ice for 30 mm to achieve a
slowly increasing pH (final pH, 12.8). One of the three tubes
was protected against the alkaline environment by adding 0.4 ml
solution of I M glucose and 14 msi �3-mercaptoethanol (solution
F) before adding solutions D and E. In this tube, the DNA did
not unwind (100% dsDNA; tube T). One of the two other tubes
exposed to the alkaline environment was vortexed until no
dsDNA was left (0% dsDNA; tube B), whereas the third tube
represented the actual measurement (tube P). The cells were
incubated at 15#{176}C,allowing the DNA to unwind. For every cell
line, an optimal unwinding time had to be determined; for the
A2780 and 22B cells, an unwinding time of 60 mm was chosen,
and for the C26-lO cell line, the unwinding time was 30 mm.
The incubation was terminated by adding 0.4 ml solution F to
tubes P and B, which were mixed subsequently and chilled on
ice. The dsDNA was stained by adding I .5 ml solution of 6.7
p.g/ml ethidium bromide and 13.3 mtvi NaOH. Fluorescence was
measured on a SPEX F]uoromax fluorescence spectrometer
(Perkin Elmer/Cetus, Norwalk, CT; excitation, 520 nm; ana-
lyzer, 590 nm). The percentage of dsDNA was calculated by:
(P - B)/(T - B) X 100%.
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Table I Summary of the sensitivity of the tested cells todFdC and CDDP
The cells were exposed continuously to the drugs for 1, 4, 24, and72 h, followed by culture in drug-free medium until 72 h after initialdrug addition. Values represent the mean IC50 ± SEM of three to eightexperiments
524 Synergistic Interaction between Cisplatin and Gemcitabine
Cell line Exposure (h) dFdC (nM) CDDP (�i.M)
A2780 1
424
72
52.5 ± 8.4
12.4 ± 2.7
4.34 ± 2.16
2.16± 1.01
10.6 ± 2.84
2.86 ± 0.59
1.20 ± 0.48
1.96 ±0.77
ADDP 14
24
72
730 ± 213
239±117
193±43
625±154
460 ± 220
197±82
63±15
52±13
AG6000 14
24
72
>106
>106
>106
50.5 ± 20.2
54.2 ± 13.817.1 ± 4.5
4.70±0.88
4.51 ± 1.01
22B I
4
24
72
62.3 ± 12.5
14.0 ± 3.5
4.50 ± 1.21
0.86 ± 0.32
56.3 ± 15.6
14.7 ± 5.8
5.83 ± 0.52
4.10 ± 0.10
C26-l0 14
24
72
655 ± 141
254 ± 10924.7 ± 9.1
22.6 ± I 1 .6
23.8 ± 3.8
6.00 ± 1.082.95 ± 0.67
3.28 ± 0.74
RESULTS
Growth Inhibition Tests. The sensitivities of the fivecell lines to dFdC and CDDP, expressed as IC50 values, are
listed in Table 1 . Drug effects were easily detectable after 72 h
of drug exposure. The fast growing human ovarian cancer cell
line A2780 and the slow growing human head and neck squa-
mous cell carcinoma cell line 22B are the cell lines most
sensitive to dFdC. However, 22B cells were less sensitive to
CDDP than A2780 cells. As expected, ADDP was the least
sensitive cell line to CDDP, and AG6000 was the least sensitive
to dFdC. Furthermore, it seemed that AG6000 and ADDP cells
were also cross-resistant to both drugs, AG6000 about 4-fold to
CDDP and ADDP about 40-fold to dFdC (24-h exposure). In
most cell lines studied, the IC50 for 72-h exposure was less than
for shorter incubation times, consistent with the known cell
cycle specificity of dFdC. However, the IC50 value of ADDP
cells for dFdC is higher at 72 h than at 24 h of incubation. The
IC50 value of AG6000 cells for dFdC for the exposure times of
1 , 4, and 24 h was greater than the highest concentration used,
io� M. The murine colon cancer cell line C26-10 seemed to
have an intrinsic resistance to both drugs compared with A2780.
Fig. 2 shows representative relative growth inhibition
curves of ADDP and AG6000 cells when exposed to dFdC and
CDDP alone and to a combination of 1 :500 for 24 h. The curve
for dFdC in ADDP cells showed a rather flat pattern, in contrast
to CDDP; the combination curve, however, was shaped simi-
larly as that for CDDP, but with a shift to the lower concentra-
tion range. The AG6000 cells were exposed to the combination
of dFdC and CDDP simultaneously. dFdC did not affect the
growth of AG6000 cells. The growth inhibition curve of
AG6000 cells exposed to CDDP was similar to that of dFdC and
CDDP together, suggesting that the growth inhibition of the
combination is only due to CDDP exposure. At concentrations
of >30 ELM, CDDP induced cell killing. In a multiple drug effect
analysis, this effect is represented by a CI of about 1 . Fig. 3
shows representative CI/FA plots of A2780 and ADDP cells
exposed to dFdC and CDDP. In A2780 cells, the combination of
dFdC and CDDP was antagonistic (CI > 1) at exposure times of
1 and 4 h. The CIs varied over a range of fractions affected
studied; at a FA of 0.25, a high CI (CI = 5-10) was found, but
lower values (CI 2-4) were observed at a FA of 0.75 (Fig. 2).
Synergistic interaction (CI < 1) between dFdC and CDDP was
found in A2780 cells at an exposure time of 24 h. In the ADDP
cell line, CI/FA curves similar to those in A2780 cells were
found. However, in ADDP cells, synergism was observed at 4,
24, and 72 h. Also, analysis of the combination of dFdC and
CDDP with isobolograms was performed. Fig. 4 shows a rep-
resentative isobologram of ADDP cells exposed to dFdC and
CDDP for 24 h. The observed position of the combination point
on the left of the connecting line between the IC50 values of
dFdC and CDDP indicates synergism. For every experiment, the
isobologram analysis revealed the same dose-response interac-
tion as the method of Chou and Talalay (29).
Table 2 summarizes the CIs of the five cell lines exposed
to the simultaneous or sequential combination of dFdC and
CDDP. Only values at a FA of 0.5 are represented. When the
cells were exposed simultaneously to both drugs, very different
patterns were observed between the cell lines, with antagonism
in A2780 and ADDP cells but synergism for 22B cells at short
exposure times. At exposure durations of 4, 24, and 72 h,
however, the combination of dFdC and CDDP was not syner-
gistic in the 22B cell line, with increasing CIs at longer exposure
periods. In the C26-l0 cell line, interaction between the drugs
was modest, with only additive to slightly antagonistic effects.
In all three ovarian cancer cell lines, preincubation with
CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and
72 h resulted in synergism or additivity for all combinations.
This is in contrast to the simultaneous exposure schedule. Even
in the AG6000 cell line, synergism was observed. In the 22B
cell line, the pattern of the CIs per exposure time has changed
compared with the simultaneous exposure. For this schedule, the
1 - and 4-h exposure times were antagonistic, and the 24- and
72-h exposure times were synergistic. In C26-lO cells, all sched-
ules of sequential CDDP followed by dFdC were antagonistic.
In all cell lines for all exposure times, dFdC preincubation
for 4 h followed by exposure to the combination of dFdC and
CDDP was synergistic. Using this schedule, the lowest CIs were
found in the ADDP cell line. Again, in the dFdC-resistant
AG6000 cells, synergism was observed. Also, for the colon
cancer cell line C26-lO, for which no synergism was observed in
the other schedules, synergism was observed in the schedule of
preincubation with dFdC followed by schedule of the combination
of dFdC and CDDP. Surprisingly, in all cell lines, the 24-h expo-
sure time revealed a lower CI than the 72-h exposure time.
Effect of CDDP on dFdCTP Accumulation. To deter-
mine whether CDDP might influence the metabolism of dFdC,
we measured the accumulation of the main metabolite of dFdC,
dFdCTP. For this purpose, cells were exposed to 0. 1 p.M and
1 �LM dFdC as a single agent or in combination with 20 p.M and
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A [dFdC](M)
I 20
I 00
10.6 10-s 10-a 10-s
BICDDPI(M)
[dFdCJ (M)
120
100
60
0
DI
a)
a)
� 0
-20
-40I 0�
100 �iM CDDP, respectively, for either 4 or 24 h. Results in four
cell lines are summarized in Fig. 5. In AG6000 cells, no
dFdCTP could be detected, consistent with its known dCK
deficiency (23); therefore, this cell line was not included in these
10-i 10.6 10� 10-
ICDDPJ (M)
studies. Despite the lower sensitivity of ADDP cells compared
with A2780 cells, this cell line showed at the 4-h exposure time
similar or greater accumulation of dFdCTP, both at 0.1 �.LM and
1 p.M dFdC. dFdCTP accumulation was similar in A2780 and
Clinical Cancer Research 525
Fig. 2 Representative growthinhibition curves of the cell lines
ADDP (A) and AG6000 (B).Cells were exposed to dFdC (+),CDDP (A), and a combination ofboth simultaneously (#{149})for 24 h.After drug exposure, both cell
lines were cultured in fresh me-
dium. Total culture time was 72h. The lines had different sigmoi-dicity expressed as the m value,which was 1.315, 1.813, and1.646 for the ADDP cells, respec-tively, and 0, 1.084, and 1.080 forthe AG6000 cell line, respec-tively.
80
-
0
DI
4) 40
a)
80
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526 Synergistic Interaction between Cisplatin and Gemcitabine
0�1
0.00 0.25
0.00 0.25
0.50 0.75
050 0.75
Fig. 3 Synergy analysis of the
I 00 interaction between CDDP anddFdC in the cell lines A2780 (A)
and ADDP (B). Cells were ex-posed to dFdC and CDDP simul-taneously for 1 h (A), 4 h (#{149}),24
h (V), and 72 hr ( + ). The valuesof the CIs are: CI > 1, antago-nism; CI = I, additivity; and CI
< 1, synergism. Values are rep-
resentative of three to five inde-
pendent experiments.
22B cells, but in the murine colon carcinoma cell line C26-lO,
dFdCTP accumulation at 0. 1 ji.M dFdC was less. Also, exposure
to I �.LM dFdC resulted in similar accumulation of dFdCTP in
A2780 and 22B cells. In contrast, at the 24-h incubation time,
the A2780 cell line showed the highest accumulation of dFdCTP
both at 0.1 and 1 p.M dFdC, whereas in ADDP cells, the lowest
accumulation was found. At 0. 1 pM dFdC, the accumulation of
dFdCTP was undetectable in C26-lO cells.
Research. on September 10, 2021. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
15
10
C-)
U-0
5
00 100 200 300 400 500
CDDP (pM)
Clinical Cancer Research 527
Fig. 4 Isobologram analysis of exposure of ADDP cells to the com-bination of dFdC and CDDP for 24 h. 1C5() values of the drugs as single
agents are plotted on the axes; when connected by a solid line, this
represents an additive effect in case the interaction would be mutuallyexclusive; when connected by a broken line, this represents an additiveeffect when the interaction would be mutually nonexclusive. The point
representing the concentrations of dFdC and CDDP resulting in a 50%growth inhibition of the combination is plotted on the left of both
connecting lines, indicating synergism.
At an exposure time of 4 h, CDDP did not increase the
dFdCTP accumulation in any of the five cell lines, neither at 0.1
�LM dFdC in combination with 20 pM CDDP nor at 1 p.M dFdC
in combination with 100 1JM CDDP. However, C26-10 cells
accumulated less dFdCTP in the presence of CDDP at 1 �LM
dFdC in combination with 100 (LM CDDP compared with dFdC
alone. Also, at an exposure time of 24 h, CDDP did not increase
accumulation of dFdCTP in C26-l0 cells. Under these condi-
tions, not only in C26-lO cells but also in A2780 cells, CDDP
decreased dFICTP accumulation. In A2780 and 22B cells,
dFdCTP concentrations could not be measured at the 24-h
incubation time due to the toxicity of the combination causing
cell death. In the more resistant ADDP and C26-lO cells,
dFdCTP accumulation could be measured at the higher concen-
trations also, but in ADDP cells, no effect was observed,
whereas in C26-lO cells, lower dFdCTP accumulation was
found.
DNA Damage. Another interaction between CDDP and
dFdC could be due to drug-induced DNA damage. This possi-
bility was evaluated with a DNA unwinding assay. Table 3
shows the relative amounts of dsDNA in A2780, 22B, and
C26-l0 cells after a 24-h exposure to 1.5 flM dFdC, 0.75 pM
CDDP, and a combination of both. Under these conditions,
dFdC alone did not cause DNA strand breaks in the A2780 and
C26-lO cells, but 20% DNA strand breaks were found in 22B
cells. After CDDP exposure, 15-25% DNA strand break forma-
tion was found in all three cell lines. However, the combination
of dFdC and CDDP did not result in an increase in DNA strand
break formation, because the effect of the combination was less
than could be expected based on an additive interaction.
Table 2 Summary of the CIs of the combination of dFdC and CDDP
Values represent CIs (non-mutually exclusive case) calculated at
the fraction affected of 0.5 of the combination of dFdC and CDDP andare means of three to five experiments; SEs were less than 30%. Cellswere exposed to the combination either simultaneously or preincubated
with CDDP for 4 h followed by a 1-, 4-, 24-, or 72-h dFdC incubation
or incubated with dFdC for 4 h followed by a combination of dFdC andCDDP for 20 or 68 h. The ratio dFdC:CDDP was 1 :500. CI > I
indicates antagonism; CI - 1 is additive; and CI < 1 is synergism.
Drug Incubationadministration (h) A2780 ADDP AG6000 22B C26-lO
CDDP and dFdC I 5.04 1 18.93 0.96 0.41 2.40
simultaneously 4 6.37 0.27 0.98 1 .33 2.5224 0.21 0.69 1.45 3.03 0.9972 1.15 0.57 1.81 13.35 1.92
CDDP 4 h I 0.06 0.59 0.86 153.24 6.50before dFdC 4 0.30 0.01 0.39 1.04 5.18
24 0.23 0.53 0.98 0.58 2.75
72 0.02 0.26 0.43 0.48 1.50
dFdC 4 h 24 0.28 0.04 0.06 0.36 0.17before dFdC 72 0.68 0.05 0.41 0.46 0.47
and CDDP
DISCUSSION
In this study, we show a definite synergism between dFdC
and CDDP on growth inhibition in a panel of five solid tumor
cell lines. The synergy is dependent on exposure duration and
sequence ofthe drugs. Exposure to the combination ofdFdC and
CDDP simultaneously was synergistic in A2780 and ADDP
cells. Preincubation with CDDP followed by dFdC was syner-
gistic in all cell lines except C26- 10 cells. Incubation with dFdC
followed 4 h later by the combination was synergistic in all cell
lines.
Mechanistic studies concentrated on dFdC metabolism,
with emphasis on accumulation of the active metabolite
dFdCTP. No consistent pattern of the effect of CDDP on
dFdCTP accumulation was observed. It is remarkable that in the
dFdC-insensitive ADDP cells, dFdCTP accumulation was corn-
parable to that of the parent cell line A2780. In contrast to other
cell lines, ADDP cells accumulated less dFdCTP with 24-h
exposure to the drug than with 4-h exposure. This early satura-
tion may explain the lower IC50 value at 4 and 24 h than at 72
h in ADDP cells. CDDP at the 4-h exposure time decreased
dFdCTP accumulation slightly in the ADDP cell line, whereas
the combination of dFdC and CDDP on growth inhibition was
synergistic. However, in C26-lO cells, CDDP decreased
dFdCTP accumulation considerably at both the 4- and 24-h
exposure times. The explanations for these phenomena are un-
clear, but possibly, CDDP inhibits dFdC uptake into cells and,
eventually, subsequent phosphorylation. Also, it was found pre-
viously that in a combination of DAC and CDDP, CDDP
decreased the cellular uptake of DAC ( 19, 35). We plan to
explore this hypothesis further in future studies.
To investigate the mechanism of interaction further, we
studied drug-induced DNA damage. In previous studies by
others, CDDP did not affect the levels of incorporated DAC into
DNA (35). Although CDDP did not inhibit the incorporation of
ara-C into normally replicating DNA, CDDP enhanced the
Research. on September 10, 2021. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
A2780 ADDP 22B C26-10
B
528 Synergistic Interaction between Cisplatin and Gemcitabine
500
a,
� 4000
(0
Lu�. 300I-C-).5
IL
.5 2000E0�
100
0
1000
8000,
a)0
*0
� 600a-I-.
I 400200
0
A
A2780 ADDP 22B C26-10Fig. 5 Effect of CDDP on the accumulation of dFdCTP in A2780,ADDP, 22B, and C26-lO cells after 4-h (A) and 24-h (B) exposures to
dFdC with or without CDDP. Values are means ± SEM of three to fiveexperiments. I, 0. 1 �M dFdC; PA, 0. 1 p.M dFdC and 20 �i.M CDDP; �,I �LM dFdC; 0, 1 Ii.M dFdC and 100 �LM CDDP.
incorporation of ara-C into DNA undergoing repair synthesis
(36). In our study, dFdC did not cause DNA damage in A2780
and C26-l0 cells, whereas CDDP induced some damage, and
only in the 22B cell line did the combination dFdC and CDDP
cause considerable DNA damage after exposure to either drug
alone. The lack of an effect of dFdC in causing DNA damage
might be related to the observation that dFdCTP incorporation
Table 3 Effect of dFdC and CDDP on cellular dsDNA
The effect of 1-h exposure to 50 p.M VP-l6 was always included asa positive control for the assay, because VP- 16 is known to produce ds
breaks.DNA damage was evaluated as percentage of dsDNA. Values are
means ± SE of percentages of dsDNA in treated compared with that inuntreated cells, which was set at 100% in each experiment. The actual
amounts of dsDNA of cells not exposed to drugs were: A2780, 86.0 ±
5.2; 22B, 88.1 ± 7.0; and C26-lO, 73.0 ± 3.3% of fluoresence of
samples not exposed to an alkaline environment.Exposure was 24 h to dFdC (I .5 nM) and CDDP (0.75 pM) as single
agents and a combination of both agents (1 .5 n�i + 0.75 p.M). Theunwinding time in the FADU assay was 60 mm for A2780 and 22B, 30
mm for C26-I0.
Drug A2780 22B C26-lO”
dFdC 102 ± 6.7 79.2 ± 4.6 97.5 ± 6.6CDDP 89.3 ± 10.4 76.2 ± 6.7 85.8 ± 3.8
dFdC+CDDP 103 ± 8.2 83.5 ± 1 1.2 87.4 ± 6.5
VP-16 25.7 ± 15.4 32.3 ± 5.7 52.1 ± 27.3
a dFdC 20 flM and CDDP 2 �J.Mas single agents and in combination.
into DNA may inhibit exonuclease activity, thereby preventing
excision of misincorporated dFdCMP (9). Interestingly, in all
cell lines, the two drugs did not cause any obvious additive
DNA damage. Actually, in the combination, DNA damage was
always less than that caused by CDDP alone, and in A2780 and
22B cells, damage was also less than that caused by dFdC alone.
Not only might this be related to inhibition of dFdC-induced
DNA damage, but dFdC incorporated into DNA also might
result in inhibition of the repair of CDDP-DNA adducts, causing
apparent stabilization of DNA. Thus, the synergistic interaction
between dFdC and CDDP might be related to reduced DNA
repair. Moreover, dFdCTP inhibits the excision repair of intra-
strand and possibly interstrand cross-links of CDDP, which may
result in synergy (37). These mechanisms might clarify why no
increased DNA damage as a result of the combination of dFdC
and CDDP was found in the FADU assay, although these events
may enhance cytotoxicity. Further evidence that DNA repair
might play a role in the interaction of cytidine analogues with
CDDP was found in studies concentrating on the role of caffeine
(38-40). This methylated xanthine is capable of sensitizing
tumor cells to cytotoxic agents, probably by inhibiting DNA
repair. In a nude mouse model with human pancreatic adeno-
carcinoma, the combination of ara-C and CDDP did not enhance
the therapeutic effect. The addition of caffeine, however, to the
combination of ara-C and CDDP resulted in an early tumor
response followed by complete tumor regression. It is of interest
that this action of caffeine was seen only in the triple combina-
tion ara-C, CDDP, and caffeine, but not when caffeine was
combined only with either ara-C or CDDP. Probably, the addi-
tion of methylated xanthines can convert the ara-C-CDDP in-
teraction to synergism, due to inhibition of DNA repair by
caffeine. In contrast to dFdC, ara-C incorporation into DNA
does not lead to inhibition of DNA repair (41). Therefore, it is
likely that the observed interactions between dFdC and CDDP
are due to reduced DNA repair, in a manner similar to the
combination of ara-C, CDDP, and caffeine. The observation that
the sequential exposure of dFdC followed by CDDP was syn-
ergistic in all cell lines supports this hypothesis. Pretreatment of
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Clinical Cancer Research 529
cells with dFdC will allow sufficient dFdCTP accumulation and
incorporation into DNA, resulting in decreased repair when
CDDP-DNA adducts are formed.
Other studies of the mechanism of synergistic interaction in
the combination of cytidine analogues, including DAC, and
ara-C, and CDDP, suggest that the synergy is due to the incor-
poration of DAC into the DNA. DNA hypomethylated by DAC
bound greater amounts of CDDP than normally methylated
DNA (19). Hypomethylation could produce conformational
changes in DNA, which make potential sites of CDDP adduct
formation more accessible. Like dFdCTP, ara-CTP does not
hypomethylate DNA. It was found that ara-C had no effect on
the binding of CDDP to DNA, the induction of CDDP-induced
interstrand DNA cross-links, the excision of CDDP-induced
interstrand DNA cross-links, or the excision of total platinum
from DNA. However, ara-C delayed the recovery of DNA
synthesis inhibited by CDDP markedly (20). Similar mecha-
nisms may operate in the combination of dFdC and CDDP. It
seems likely that one site of interaction between dFdC and
CDDP is at the DNA level, because in AG6000 cells, which did
not accumulate dFdCTP, no synergism is observed with simul-
taneous exposure. The sequential effects of the combination, not
only in this cell line, but also in the other cell lines, suggest an
additional type of interaction between dFdC and CDDP not
necessarily requiring dFdC phosphorylation. The synergistic
effect of both sequences suggests not only an effect of dFdC (or
its metabolites) on CDDP uptake and intracellular distribution,
but also an effect of CDDP on dFdC metabolism. One such
interaction may be at the level of regulation of dFdC metabo-
lism. CDDP is known to inhibit ribonucleotide reductase (21),
which may lead to a decrease of dCTP and an imbalance in other
deoxyribonucleotides. This cannot only influence dFdC metab-
olism; it also affects DNA repair. Furthermore, differences in
enzyme parameters (Vmax and Km for ribonucleotide reductase
and DNA repair enzymes) in different cell lines might clarify
the difference in the interaction between dFdC and CDDP in the
cell lines (42). It remains to be investigated whether dFdCTP
affects CDDP, including adduct formation, or whether CDDP
affects dFdC. Both types of interaction may occur.
The combination of dFdC and CDDP is synergistic in vitro.
This synergism is time and schedule dependent. The most ef-
fective sequence is 4-h dFdC preincubation followed by the
combination of dFdC and CDDP, which was synergistic in all
cell lines. This indicates an enhanced effect of dFdC on CDDP
due to this preincubation. This sequential synergistic interaction,
in addition to nonoveriapping toxic side effects, is of interest for
future clinical studies, especially in tumor types such as non-
small cell lung cancer, ovarian cancer, and head and neck
cancer, in which both drugs have shown single agent activity.
ACKNOWLEDGMENTS
We thank Dr. S. Ackland (University of Newcastle, Australia) for
critical reading of the manuscript.
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