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Experimental Therapeutics, Preclinical Pharmacology |
Pharmacology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan [F. Ka., F. Ko., Y. K., T. N., Y. T., H. F., K. N.]; Radiation Oncology Division, National Cancer Center Hospital, Tokyo 104-0045, Japan [N. S.]; and Discovery Research Laboratories, Shionogi & Co., Ltd., Osaka 553-0002, Japan [T. Y.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we examined whether combination chemotherapy with NDP and VP-16 resulted in as marked a synergistic anticancer activity against lung cancers as that of the CDDP and VP-16 combination. We also selected CPT-11 as the best candidate drug for combination with NDP using a three-dimensional analysis model established by our group.
Because our final goal is to eventually develop the combination for use in a clinical setting, we attempted to identify the optimal treatment schedule that must be used to achieve the synergistic interaction and to determine the biochemical mechanism underlying the synergistic interaction between the two drugs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Culture.
Human SCLC cell line SBC-3 and human NSCLC cell line PC-14 were used.
They were kindly provided by Dr. I. Kimura (Okayama University,
Okayama, Japan) and Dr. Y. Hayata (Tokyo Medical College, Tokyo,
Japan), respectively. Both cell lines grew as very loosely adherent
floating aggregates in small clumps. The cells were propagated in RPMI
1640 supplemented with 10% heat-inactivated FCS, 100 µg/ml
streptomycin, and 100 units/ml penicillin in an incubator at
37°C and 100% humidity with 5% CO2 and air,
as described previously (16)
.
Antiproliferative Activity.
We measured the inhibition of cell proliferation after drug treatments
as the antitumor activity using a regrowth assay described previously
(16)
. Briefly, cells were exposed to drugs alone or in
combination for 6 days at 37°C in an atmosphere of 100% humidity
with 5% CO2; the cells were then pipetted six to
eight times until almost all cells appeared as single cells and counted
with a TOA Microcell counter (CC-108; TOA Medical Electronics Co.,
Kobe, Japan). For each drug, concentration-effect curves were drawn as
plots of the fraction of surviving cells (unaffected cell fraction,
fu) versus drug
concentration.
The cell proliferation ratio of the treated:control cultures (T:C%) was calculated as follows: [(the number of treated cells on day 6)/(the number of treated cells on day 0)]/[(the number of control cells on day 6)/(the number of control cells on day 0)] x 100%. The IC50 was defined as the drug concentration required for a 50% reduction in the number of cells. Four or five independent experiments were carried out for each.
To check the effect of the drug treatment schedule on the effect of the
combination, the cells were treated either by simultaneous exposure to
the two drugs or by sequential exposure to NDP followed by CPT-11
(NDP
CPT-11) and vice versa (CPT-11NDP) for 3 h.
For the sequential exposure treatment, cells were exposed to the first
drug for 3 h, washed in fresh medium once, and then immediately
exposed to the second drug for 3 h. The treated cells were
cultured in drug-free medium until evaluation.
Assessment of the Effect of the Combination.
The theoretical basis of three-dimensional model analysis has been
described in a previous study (17)
. Briefly, according to
Chou and Talalay (18)
, the theoretical additive effect can
be calculated from
(fa)A and
(fa)B, which are
the fractions affected by drug A and drug B, respectively.
The calculated response surface
(SA,B)cal was obtained by
integrating the theoretical additive effects with respect to
(fa)A and
(fa)B given by
Eq. A.
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The 95% confidence limit values were obtained to multiply the
variances SA2 and
SB2 of drug A and drug B,
respectively, and were integrated with respect to the drug
concentrations in accordance with the following equation to obtain the
95% confidence limit surface,
(SA,B)CL.
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df is the
t value (df = n – 1; = 0.05),
a is the concentration of drug A(k -
l), and b is the concentration of drug
B(m - n). Dividing the raw combination effect surface by the 95% confidence limit surface, regions >1 or <-1 are defined as synergism or antagonism, respectively. The region between 1 and -1 represents additive effect.
Preparation of Nuclear Extracts.
Crude nuclear extracts were prepared as described previously by
Deffie et al. (19)
. In brief, cells were
collected by centrifugation, washed twice with cold NB, resuspended in
1 ml of cold NB, and 9 ml of cold NB containing 0.35% (v/v) Triton
X-100 and 1 mM phenylmethylsulfonyl fluoride
were added. The cell suspension was kept on ice for 10 min and
washed with Triton X-100-free cold NB, and the nuclear protein was
eluted for 1 h at 4°C with cold NB containing 0.35
M NaCl. A nuclear protein solution was obtained
by centrifugation at 18,000 x g for 10 min, and its
protein concentration was determined using the method of Bradford
(20)
with bovine plasma 
-globulin as the standard.
DNA Topoisomerase I Activity.
The activity of DNA topoisomerase I was determined by measuring
the relaxation of supercoiled Escherichia coli DNA (pBR322)
essentially as described by Liu and Miller (21)
.
For measurement of the total topoisomerase I activity in SBC-3 cells,
the reaction mixtures used contained 100 mM KCl,
10 mM MgCl2, 1
mM DTT, 0.1 mM EDTA, 10%
(v/v) glycerol, 50 mM Tris-HCl (pH 7.4), 0.7 µg
of pBR322, and crude nuclear extract. The reaction mixtures used for
measuring the inhibition of DNA relaxation by topoisomerase I
inhibitors comprised the specified amounts of the nuclear extract (1.0
µg/ml protein) and drug solution or the equivalent volume of water in
addition to the above-mentioned components. The reaction mixtures were
incubated at 37°C for 10 min, and the reactions were terminated by
adding 45 µl of a dye solution containing 2.5% (w/v) SDS, 0.01%
(w/v) bromphenol blue, and 50% (v/v) glycerol. The mixtures were
applied to 0.7% (w/v) agarose gel and electrophoresed for 4 h
with a running buffer of Tris-acetate EDTA. The gel was stained with 2
mM ethidium bromide and photographed under
transillumination with 300 nm UV light.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the dose-response curves for the growth of SBC-3 cells. NDP
inhibited the proliferation of SBC-3 cells by 98%, 93%, 75%, 54%,
27%, 6%, and 2% at a concentration of 0.005, 0.01, 0.025, 0.05, 0.1,
0.25, and 0.5 µg/ml, respectively. Consequently, its
IC50 value for growth inhibition of SBC-3 cells
was 0.053 µg/ml, which is equivalent to 0.18 µM, which
was almost the same as that of CDDP. The IC50
values of CPT-11, VP-16, ADM, and VDS for the growth inhibition of
SBC-3 cells were 0.042, 0.025, 0.0027, and 0.00028 µg/ml,
respectively.
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. The
IC50 values of NDP, CDDP, and CPT-11 for the
growth inhibition of PC-14 cells were 0.61, 0.40, and 0.85 µg/ml,
respectively.
We chose the one- or two-log dose range that included the
IC50 values for the combination effect study
(Table 1)
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, A–G, which illustrate the occurrence of complex drug-drug
interactions with a concentration dependence. Fig. 2
A shows
the effects of the combination of CDDP with the topoisomerase II
inhibitor VP-16 in SBC-3 cells, and the concentration dependence of the
drug-drug interaction as well as the complexity of the interaction is
demonstrated clearly. The region of approximately 0.05–0.25 µg/ml
CDDP and 0.025–0.1 µg/ml VP-16, depicted in blue,
represents a statistically significant synergic interaction. The effect
of the combination at nadir concentrations of both drugs was observed
to be less than additive. On the whole, these findings were consistent
with those of a previous study (17)
.
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B). The pattern of the concentration
dependence of the drug-drug interaction was also similar to that for
the combination of CDDP and VP-16. That is, the effect of the
combination of NDP and VP-16 is biphasic and depends on the
concentrations of both drugs.
To determine the role of the effect on topoisomerases as the cytotoxic
mechanism of VP-16 on the synergistic interaction, we further
investigated the effects of combinations of NDP + ADM, CPT-11, and
other drugs. Fig. 2
C shows the effect of the combination of
NDP and the topoisomerase II inhibitor ADM (22)
. A
synergistic interaction was observed within a limited dose range, such
as 0.005 µg/ml NDP and 0.0005–0.001 µg/ml ADM. On the other hand,
an additive effect was seen with NDP concentrations of more 0.01
µg/ml, regardless of the ADM concentration.
A remarkable synergistic interaction was observed between NDP and the
topoisomerase I inhibitor CPT-11 at concentrations of approximately
0.005–0.05 and 0.005–0.1 µg/ml, respectively (Fig. 2
D).
Regions depicted in blue represent statistically significant
synergic interactions. An additive effect, without any relation to the
CPT-11 concentration, was noted at higher concentrations (>0.1
µg/ml) of NDP. A similar tendency was also observed with the CDDP and
CPT-11 combination (Fig. 2
E). In other words, a synergistic
interaction was seen in combinations of low concentrations of both
drugs, whereas an antagonistic interaction was noted at comparatively
high concentrations.
The three-dimensional graph of the NDP and VDS combination showed that
this combination has a less than additive effect (Fig. 2
F).
VDS is known to be a tubulin modulator but not a topoisomerase I or II
inhibitor. A similar interaction was also observed in the NDP and CDDP
combination (Fig. 2
G). Both drugs are known to exert DNA
damage but do not affect topoisomerases. These results suggest that a
topoisomerase inhibitor, particularly CPT-11, is most likely the best
candidate for combination with NDP.
Effects of Combinations of CPT-11 and NDP or CDDP in PC-14 Cells.
Fig. 3
, A and B, presents the three-dimensional graphs
of the combinations of CPT-11 and NDP or CDDP, respectively, in NSCLC
PC-14 cells. A remarkable synergistic interaction was also observed
even when NDP and CPT-11 were combined at concentrations of <0.5 and
<1.0 µg/ml, respectively. In the combination of CDDP and CPT-11, the
drug interaction pattern was biphasic and depended on the drug
concentrations. A synergistic interaction was observed at lower drug
concentrations, whereas an antagonistic effect was observed at higher
concentrations.
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Schedule-dependent Synergism between NDP and CPT-11.
The synergy between these drugs seems to be affected by the drug
treatment schedule because of their differing mechanisms. We sought to
explore the influence of the drug treatment schedule on the effect of
the combination to identify the most suitable schedule. In this
experiment, 3 h was chosen as the exposure period because both NDP
and CPT-11 disappeared within 3 h after administration to patients
as short i.v. infusions. Table 1
shows the IC50
values of NDP and CPT-11 when SBC-3 cells were treated with the two
drugs for 3 h. Cells were simultaneously exposed to both drugs for
3 h or sequentially exposed to CPT-11 for 3 h and then
exposed to NDP for 3 h (or vice versa) at
concentrations near their IC50 values.
Fig. 4
illustrates the schedule-dependent synergism between NDP and CPT-11. A
remarkable synergistic interaction was observed when the cells were
simultaneously exposed to NDP and CPT-11 at concentrations of 0.25–5.0
and 0.5–5.0 µg/ml, respectively, for 3 h. The magnitude of
synergism seen in the brief drug exposure experiment was similar to
that in the continuous drug exposure experiment (Fig. 3
D).
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B). A similar tendency was also observed with sequential
exposure to NDP followed by CPT-11, with no synergistic effect induced
at restricted doses (Fig. 4
C). Results such as complete loss
of potentiation on sequential exposure to CPT-11 followed by NDP and
the lower degree of synergism observed on sequential exposure to NDP
followed by CPT-11 imply that the synergistic interaction between both
drugs is schedule dependent.
Effect of NDP on the Inhibitory Effect of SN-38 on Topoisomerase I.
To analyze the synergistic interaction between the drugs, we examined
the effect of NDP and SN-38 (the active form of CPT-11) on the
activities of DNA topoisomerase I in the nuclear protein extract of
SBC-3 cells.
First, the topoisomerase I activity in control SBC-3 cells was measured
by the relaxation assay in a serial dilution and compared with that in
cells pretreated with NDP or CDDP at 0.25 and 2.5 µg/ml,
respectively, for 3 h. The supercoil pBR322 DNA was completely
relaxed with 0.1 µg of the nuclear protein extract from SBC-3 cells
and partially relaxed with 0.01 and 0.001 µg of the nuclear protein
extract from SBC-3 cells, and the relaxation activity did not change
after pretreatment with NDP, as shown in Fig. 5
A. The inhibitory effect of SN-38 on the catalytic activity
of topoisomerase I from SBC-3 cells treated with NDP was also compared
with that on the enzyme from control cells. The inhibitory activity of
SN-38 was similar in both and was the concentration-dependent, as shown
in Fig. 5
B. These results confirm that neither the catalytic
activities of topoisomerase I nor its susceptibility to topoisomerase I
inhibitors was affected by pretreatment with NDP.
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C, 0.1 µg of the
nuclear protein extract from SBC-3 cells relaxed the supercoiled pBR322
DNA, and this relaxation was inhibited by treatment with SN-38 at 1.0
µg/ml, but not by treatment with SN-38 at <0.3 µg/ml. In
the presence of NDP (25 µg/ml), on the other hand, 0.03 µg/ml SN-38
was sufficient to inhibit the DNA relaxing activity of topoisomerase I.
The inhibiting effect of NDP on the SN-38-induced inhibition of
topoisomerase I was also observed in PC-14 cells (data not shown).
These results suggest that in vitro, the effect of the
combination of NDP and CPT-11 correlated with the effect of NDP on the
CPT-11-induced inhibition of topoisomerase I. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, whose object was to seek drugs superior to VP-16 for combination with NDP, it was demonstrated that a NDP and CPT-11 combination chemotherapy resulted in synergism against human lung cancer and that the effect of the combination of NDP and CPT-11 was superior to the effect of the combination of CDDP and VP-16. It is worth in clinically to substitute CDDP with non-nephrotoxic NDP in combination with CPT-11.
The three-dimensional graphs also demonstrated previously that a synergistic interaction was observed for the CDDP and VP-16 combination at higher concentrations of both drugs (17) . This is in contrast to the synergistic interaction between NDP and CPT-11 at lower concentrations in this study. These concentrations, namely, 0.005–0.01 µg/ml for NDP and 0.025–0.01 µg/ml for CPT-11, are clinically relevant doses.
CPT-11 inhibits topoisomerase I-mediated DNA relaxation through the formation of stable topoisomerase I-DNA cleavable complexes, regardless of the cell line examined (24, 25, 26, 27) . The inhibitory activity of CPT-11 on topoisomerase I is correlated with its antitumor activities (28) . To analyze the biochemical mechanism underlying the synergistic interaction between the NDP anbd CPT-11, the effects of NDP on the SN-38-induced inhibition of topoisomerase I were examined. The topoisomerase I-inhibitory activity of SN-38 was enhanced 10-fold by coexposure to NDP. These biochemical interactions might be responsible for the synergistic interaction between NDP and CPT-11 and are also thought to be a common underlying mechanism for the synergistic interaction between platinum analogues and topoisomerase I inhibitors (15) . Furthermore, the concentration of NDP required to enhance the activity of CPT-11 was 25 µg/ml, which is lower than that of CDDP (150 µg/ml; Ref. 29 ). This is sufficient to explain the greater synergism observed between NDP and CPT-11 as compared with CDDP and CPT-11. The cytotoxicity of platinum analogues such as CDDP is believed to be due to the formation of DNA adducts, which include DNA protein cross-links, DNA mono-adducts, and interstrand and intrastrand DNA cross-links (30) . Furthermore, X-ray diffraction of the cross-linked dinucleotide cis-Pt(NH3)2(d(pGpG)) has revealed that the two guanines are completely destacked and that the deoxyribose sugar of the 5'-deoxyguanosine is in a C3'-endo pucker (29 , 31) . Thus, the intrastrand platinum cross-link produces a severe local distortion in the DNA double helix, leading to unwinding and linking, which may modulate the stability of topoisomerase I-drug-DNA cleavable complexes.
The opposite results observed for CDDP + VP-16 and NDP + CPT-11 with regard to the drug concentrations inducing the synergistic interaction seem to be due to differences in the cytotoxic mechanisms between CPT-11 and VP-16 rather than NDP and CDDP because both NDP and CDDP are known to form DNA adducts and lead to cell death as a common cytotoxic mechanism (30) , whereas both CPT-11 and VP-16 are well known inhibitors of different targets, topoisomerase I and II, respectively. However, there is no evidence of any determinant being due to concentration of CPT-11 or VP-16 on the synergistic interaction in literature.
Study of treatment schedules for NDP and CPT-11 demonstrated that a remarkable synergistic interaction was produced by concurrent exposure to NDP and CPT-11; in contrast, sequential exposure treatments led to only an additive effect. These results provided important preliminary information for a future in vivo study.
Potent in vitro and in vivo cytotoxicity, relatively low neurotoxicity and nephrotoxicity, and a large in vivo bioavailability have ensured the position of NDP as a promising platinum analogue for further clinical development as a salvage and primary chemotherapeutic agent for the treatment of patients with advanced lung cancer. Also, high clinical response rates of lung cancer to CPT-11 have been reported previously (32) . Based on these findings, the combination of NDP and CPT-11 seems to be a very promising one for cancer chemotherapy. Further examination of these drug interactions in in vivo preclinical studies should provide useful information for future combination chemotherapy regimens.
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid for Cancer
Research and grants from the Second Term Comprehensive 10-Year Strategy
for Cancer Control; the Ministry of Health and Welfare of Japan; the
Education, Science, Sports and Culture of Japan; and a
government-supervised trust fund established by the Shionogi and Co.,
Ltd. (Osaka, Japan). 
2 To whom requests for reprints should be
addressed, at Pharmacology Division, National Cancer Center Research
Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan. Phone:
81-3-3542-2511; Fax: 81-3-3542-1886; E-mail: fkanzawa{at}gan2.res.ncc.go.jp
3 The abbreviations used are: CDDP, cisplatin;
VP-16, etoposide; NDP, nedaplatin; CPT-11, irinotecan; VDS, vindesine;
ADM, Adriamycin; SN-38, 7-ethyl-1-hydroxy-CPT; SCLC, small cell
lung cancer; NSCLC, non-small cell lung cancer; NB, 2 mM
KH2PO4, 5 mM MgCl2, 150
mM NaCl, 1 mM ethyleneglycol bis(b-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, and 1
mM DTT (adjusted to pH 6.5).
Received 6/27/00; revised 10/23/00; accepted 10/24/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), CPT-11 (•), VP-16 (
), VDS (
),
ADM (
), and CDDP (
) at various concentrations for 6 days, and
then the cell proliferation ratio of the treated:control cultures was
calculated. Antitumor activities were evaluated as described in
"Materials and Methods." Each data point represents the mean from
four or five independent experiments.
