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Experimental Therapeutics |
Divisions of Medical Oncology [C. E., K. C. B.] and Oncology Research [S. A. B., C. G. H., R. S., S. H. K.] and Department of Laboratory Medicine [X-Y. W., C. D. J.], Mayo Clinic and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School [K. C. B., S. H. K.], Rochester, Minnesota 55905; University of Maryland Greenebaum Cancer Center and Baltimore Veterans Medical Center, Baltimore, Maryland 21201 [D. D. R.]; Department of Pathology, Free University Hospital, 1081 HV Amsterdam, the Netherlands [G. L. S.]; and Division of Experimental Therapy, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands [M. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The activity of these agents reflects their ability to target topo I, a nuclear enzyme involved in transcription and replication (7, 8, 9) . SN-38, the active metabolite of irinotecan, and other CPT derivatives stabilize covalent complexes between topo I and nuclear DNA (10, 11, 12) . When moving replication complexes encounter these topo I-DNA complexes, DNA double-strand breaks result (7 , 13 , 14) . In response to these double-strand breaks, cells initially accumulate in the S phase of the cell cycle (15, 16, 17) . This arrest is followed by DNA repair or induction of apoptosis (18, 19, 20, 21) .
Sensitivity of cells to topo I poisons is modulated by a variety of factors, including topo I content, cell cycle distribution, and DNA repair capability (reviewed in Refs. 7 , 14 , 22 , and 23 ). In addition, sensitivity to CPT derivatives appears to be regulated by drug accumulation. Previous studies have demonstrated that accumulation of TPT and SN-38 is decreased slightly in cells that overexpress P-glycoprotein (24, 25, 26, 27) . Higher levels of resistance to SN-38 and TPT have been observed in cell lines that were selected for mitoxantrone resistance (28, 29, 30, 31) and subsequently shown to overexpress BCRP, a member of the ATP binding cassette family of transporters (31 , 32) . Although these observations raise the possibility that BCRP might modulate accumulation and cytotoxicity of TPT and SN38, experiments that directly address this hypothesis, e.g., by analyzing drug accumulation and cytotoxicity in BCRP-transfected cell lines, have been limited. Whether BCRP modulates drug sensitivity in unselected human lines is also unknown. Moreover, the ability of BCRP modulators to alter sensitivity to SN-38 or TPT has not been extensively explored.
CI1033 (Fig. 1)
is a quinazoline-based HER family tyrosine kinase inhibitor that is
currently being evaluated as a potential anticancer agent
(33)
. The HER family of receptors includes EGFR, HER-2,
HER-3, and HER-4. Over the last decade, considerable evidence has
implicated this receptor tyrosine kinase family in the development and
progression of a variety of human tumors (34
, 35)
. The
founding member of this family, EGFR, is commonly amplified and mutated
in high-grade glioblastomas (36)
. HER-2 is frequently
amplified and overexpressed in breast cancer (37
, 38) ,
often in conjunction with elevated EGFR. Overexpression of these
receptors is associated with antiestrogen resistance and poor prognosis
of breast cancer (37
, 38)
. Coexpression of EGFR and HER-2
has also been associated with shortened survival of patients with
carcinomas of the prostate, ovary, and upper aerodigestive tract
(39, 40, 41)
. These observations have prompted extensive
efforts to target HER family members using monoclonal antibodies
(37
, 42)
or small molecule inhibitors of receptor tyrosine
kinases (43
, 44)
.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Buffers.
Medium A contained MEM, 10% heat-inactivated FBS, 1 mM
sodium pyruvate, 100 units/ml penicillin G, 100 µg/ml streptomycin,
and 2 mM glutamine. Buffer B consisted of serum-free RPMI
1640 containing 10 mM HEPES (pH 7.4). Cell lysis buffer
(54)
consisted of 6 M guanidine hydrochloride,
250 mM Tris-HCl (pH 8.5 at 21°C), and 10 mM
ETDA supplemented immediately before use with 1 mM
-phenylmethylsulfonyl fluoride (added from a 100-fold stock in
anhydrous isopropanol) and 1% (v/v) ß-mercaptoethanol.
Clonogenic Assays.
T98G, HCT8, MCF-7, PC-3, and Ovcar-3 cells were obtained from the
American Type Culture Collection (Manassas, VA). For clonogenic assays,
trypsinized T98G cells were diluted with medium A, plated in 35-mm
tissue culture plates (500 cells/plate), and incubated for 14–16 h to
allow cells to adhere. Drugs were added to the indicated final
concentrations from 1000-fold concentrated stocks. Control plates
received the corresponding volume of diluent. After a 24-h incubation,
plates were washed twice with serum-free MEM, refed with medium A, and
incubated for 10 days. The resulting colonies were stained with
Coomassie Brilliant Blue so that visible colonies could be counted.
Control plates typically contained 150–200 colonies. An identical
approach was used with other cell lines, except that the number of
cells plated was adjusted to 1300 (PC-3) or 2000 (Ovcar-3) to achieve
150–200 colonies in control plates, and medium A was replaced by
medium consisting of RPMI 1640-5% heat-inactivated FBS (HCT8); Ham’s
F-12 medium-7% heat-inactivated FBS (PC-3); MEM containing Earle’s
salts, 10% heat-inactivated FBS, nonessential amino acids, 1
mM sodium pyruvate, and 10 µg/ml insulin (MCF-7); or MEM
containing 20% heat-inactivated FBS (Ovcar-3), each of which was
supplemented with 100 units/ml penicillin G, 100 µg/ml streptomycin,
and 2 mM glutamine.
To examine the effect of sequencing, cells were plated as described above, incubated with one drug for 24 h, washed twice, incubated with the second drug for 24 h, washed, and incubated in drug-free medium for the duration of the 7–14-day incubation period. In these experiments, exposure to single agents took place at the same time as exposure to the drugs in sequence.
BCRP Transfectants.
MDA-MB-231 cells were transfected with pcDNA3 encoding BCRP behind the
constitutive cytomegalovirus promoter or with empty vector using
techniques described previously (55
, 56)
. Clones selected
in Geneticin were maintained in 400 µg/ml Geneticin until they were
subjected to colony-forming assays and drug accumulation studies as
described above.
Analysis of Combined Drug Effects.
Combined drug effects were analyzed by the median effect method
(57)
. In brief, cells were treated with serial dilutions
of each drug individually and with both drugs simultaneously or
sequentially at a fixed ratio of doses that typically corresponded to
one-half, five-eighths, three-fourths, seven-eighths, 1.0, and 1.5
times the individual IC50 values. The fractional survival
(f) was calculated by dividing the number of colonies in
drug-treated plates by the number of colonies in control plates. Log
[(1/f) - 1] was plotted against log [drug
dose]. From the resulting graphs, the x intercept (log
IC50) and slope m were calculated for each drug
and for the combination by the method of least squares and
then used to calculate the doses of the individual drugs and the
combination required to produce varying levels of cytotoxicity
according to the following equation.
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Unless otherwise indicated, experiments were repeated until three
replicates yielded correlation coefficients of 
0.9 for all three
median effect lines. Results of multiple experiments are summarized by
indicating the mean ± SD of the CI at the indicated
level of colony inhibition.
Alkaline Elution.
Logarithmically growing cells were labeled for 24 h with 1
µM [14C]thymidine, sedimented, and
incubated for 1–2 h at 37°C in fresh medium A. To measure
steady-state levels of topo I-mediated DNA single-strand breaks,
aliquots were then incubated for 30 min at 37°C with the indicated
concentrations of SN-38, diluted with ice-cold 75 mM
NaCl-2.4 mM EDTA (pH 7.4 at 4°C), and deposited on
Nucleopore phosphocellulose filters (1 µm, pore size; VWR Scientific,
Minneapolis, MN) by gentle suction. All additional steps were performed
as described recently (53)
. In brief, cells were lysed by
allowing 5 ml of buffer consisting of 1% (w/v) SDS, 100 mM
glycine, 25 mM EDTA (pH 10), and 0.5 mg/ml proteinase K to
drip through the filters. After filters were washed with 20
mM EDTA (pH 10), DNA was eluted with 20 mM EDTA
(adjusted to pH 12.1 with tetrapropylammonium hydroxide). The
distribution of radiolabel in the eluate, the tubing of the elution
apparatus, and the filter were analyzed as described by Covey et
al. (58)
. Cells that received 150–900 cGy of
-irradiation from a 137Cs source were included in each
experiment as a standard curve.
Immunoblotting.
Whole cell extracts were prepared by lysing washed cells in cell lysis
buffer. After a 4-h incubation at 20°C, samples were sonicated to
shear the viscous DNA, treated with iodoacetamide to block free
sulfhydryl groups, dialyzed at 4°C into 4 M urea and then
into 0.1% (w/v) SDS (59)
, and lyophilized to dryness.
Subsequent electrophoresis and immunoblotting were performed as
described previously (60)
. Alternatively,
detergent-soluble protein extracts for EGFR blotting were isolated by
incubating cells at 4°C for 15 min in extraction buffer consisting of
150 mM NaCl, 20 mM HEPES (pH 7.5), 1.5
mM MgCl2, 10 mM EGTA, 1
mM sodium vanadate, 10% (v/v) glycerol, and 1% (v/v)
Triton X-100 supplemented with 2 µg/ml aprotinin, 5 µg/ml
leupeptin, 50 µg/ml
N-p-tosyl-L-lysine chloromethyl
ketone, 100 µg/ml
N-p-tosyl-L-phenylalanine
chloromethyl ketone, 100 µg/ml -phenylmethylsulfonyl fluoride, and
50 µg/ml soy bean trypsin inhibitor. After particulate material was
removed by centrifugation at 11,000 x g for
15 min at 4°C, protein concentrations in the supernatant were
estimated (61)
. Aliquots containing 80 µg of protein
were diluted with an equal volume of loading buffer [63 mM
Tris-HCl (pH 6.8 at 21°C), 2% (w/v) SDS, 10% (v/v) glycerol, and
0.005% (w/v) bromphenol blue], heated to 95°C for 5 min, and
applied to SDS-polyacrylamide gels containing a 4–20% acrylamide
gradient. Separated proteins were electrophoretically transferred to
nitrocellulose. Membranes were incubated for 1 h at 21°C in
blocking buffer consisting of 5% nonfat dry milk, 137 mM
NaCl, 20 mM Tris-HCl (pH 7.6 at 21°C), and 0.1% (w/v)
Tween 20. Filters were subsequently incubated overnight at 4°C with 1
µg/ml anti-EGFR or anti-phosphorylated EGFR antibody in blocking
buffer, washed, and incubated for 1 h with peroxidase-conjugated
secondary antibody. After additional washes, bound antibody was
detected by enhanced chemiluminescence.
Band Depletion Assay.
Replicate plates of cells were treated with 8 µM CI1033
or diluent in medium A for 1 h. After SN-38 was added to a final
concentration of 0–30 µM, samples were incubated for an
additional 45 min. Proceeding one plate at a time, cells were rapidly
washed twice with ice-cold buffer B and immediately solubilized by
addition of 3 ml of cell lysis buffer followed by agitation. As
described previously, the rapid denaturation in this guanidine
hydrochloride-containing buffer traps covalent topo I-DNA complexes
(60)
. Subsequent preparation for electrophoresis and
immunoblotting was performed as described above.
HPLC Analysis of SN-38 or CI1033 Accumulation.
Twenty 100-mm plates of cells were grown to 70% confluence for each
data point. To assess the effect of CI1033 on SN-38 accumulation, T98G
or BCRP-transfected MDA-MB-231 cells were treated with diluent or 8
µM CI1033 for 1 h. SN-38 was then added to a final
concentration of 1 µM for 35 min. The cells were
trypsinized in the continued presence of the drugs, centrifuged at
100 x g at 4°C for 10 min, washed twice
with ice-cold calcium- and magnesium-free Dulbecco’s PBS, and lysed in
1 ml of -20°C methanol. The precipitate was sedimented and analyzed
for protein as described previously (62)
. Aliquots of the
supernatant (100 µl) were treated with 1 µl of concentrated
phosphoric acid to convert all SN-38 to the lactone form, which was
then analyzed on a Beckman (Palo Alto, CA) model 125 dual-pump gradient
HPLC equipped with a temperature-regulated model 507e autosampler,
model 168 diode array detector, and an IBM personal computer 350 with
Beckman Gold Nouveau software. A Brownlee MPLC Newguard C18 precolumn
(3.2 mm x 15 mm x 7 µm) and a Beckman
Ultrasphere ODS column (4.6 mm x 250 mm x 5 µm) were preequilibrated for 20 min with mobile phase A
[0.05% (v/v) triethylamine in H2O (pH 4.0 with acetic
acid)]. Separation was accomplished by applying the sample in mobile
phase A and eluting with 100% mobile phase A for 10 min followed by a
linear gradient from 0–20% MeOH over 10 min and a linear gradient
from 20–100% MeOH over the next 10 min. Detection was at 384 nm,
although the spectrum of the identified peak was also compared with
that of bona fide SN-38 using a diode array detector. An
identical approach was used to examine CI1033 accumulation in BCRP- or
empty vector-transfected MDA-MB-231 cells.
Flow Cytometry.
For cell cycle analysis, log phase T98G cells were incubated for
24 h with the indicated concentrations of SN-38 or CI1033, washed,
and incubated in drug-free medium A for 0–48 h. At the completion of
the incubation, cells were released by trypsinization, sedimented at
200 x g for 10 min, washed twice with
ice-cold calcium- and magnesium-free Dulbecco’s PBS, and fixed by
dropwise addition of ethanol to a final concentration of 50% (v/v).
Subsequent digestions with RNase A, staining with propidium iodide, and
flow microfluorimetry were performed as described previously
(63)
.
To evaluate TPT uptake and efflux, cellular TPT content was analyzed on a Becton Dickinson FACScan (San Jose, CA) using an excitation wavelength of 488 nm and an emission wavelength of 585 nm, as described previously (25 , 28) . Various cell lines grown to 50–60% confluence in 100-mm dishes were incubated for 1 h with diluent or 8 µM CI1033, trypsinized in the continued presence of diluent or CI1033, sedimented at 100 x g for 6 min, and resuspended in medium A containing 10 mM HEPES (pH 7.4) and diluent or CI1033. For uptake studies, cells were subjected to flow microfluorometry at varying intervals during the first 3 min after the addition of 20 µM TPT. For efflux studies, cells were incubated with 20 µM TPT for 8 min to assure steady-state uptake, diluted 10-fold with medium A containing 10 mM HEPES (pH 7.4), and examined at various time points during the first 3 min after dilution.
RT-PCR.
Polyadenylated RNA (1 µg) from semiconfluent T98G, HCT8, or PC-3
cells was reverse transcribed with random hexamers and 2 units of avian
myeloblastosis virus reverse transcriptase (Promega, Madison, WI) under
standard conditions (64)
. One-twentieth of the cDNA
product was used for each amplification. PCR reactions were performed
in 50-µl volumes containing 50 mM KCl, 10 mM
Tris-HCl (pH 8.3 at 20°C), 2.5 mM MgCl2, 0.4
µM of each primer, 400 µM dNTPs, and 5
units of Taq polymerase. Reactions were amplified for a total of 33
cycles on a Perkin-Elmer DNA thermal cycler using 94°C for
denaturation (1 min), 55°C for annealing (1 min), and 72°C for
extension (2 min), followed by two cycles in which the extension step
was lengthened to 7 min. The primer pairs used were
5'-TGTTTGGAAGGTCCGGGTGA-3' and 5'-GTCCCAGGATGGCGTTGAGA-3' for BCRP
(amplify a fragment of 382 bp) or 5'-ACGTTATGGATGATGATATCGC-3' and
5'-CTTAATGTCACGCACGATTTCC-3' for ß-actin (amplify a fragment of 644
bp). After amplification, 40% of the product was separated on a 1.2%
(w/v) agarose gel in TAE buffer, stained with 0.5 µg/ml
ethidium bromide, and photographed under UV light. Sequencing confirmed
that the RT-PCR products corresponded to BCRP and ß-actin
transcripts.
Miscellaneous Methods.
To assess the effect of CI1033 on topo I activity, log phase T98G cells
in medium A were treated with 8 µM CI1033 or 0.1% DMSO
for 60 min at 37°C. Nuclear extracts were then prepared and assayed
for topo I activity (ability to relax supercoiled plasmid) as described
previously in detail (25
, 65)
. To evaluate the effect of
CI1033 on topo I localization, cells treated for 1 h with 8
µM CI1033 were fixed and stained with C-21 anti-topo I
antibody followed by rhodamine-conjugated goat antimouse IgM as
described previously (66)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 
). Exposure to CI1033 alone resulted in an IC50 of
3.7 ± 0.7 µM (n = 8; Fig. 2B
, ). Treatment with both agents
simultaneously for 24 h (Fig. 2, A and B
,
•) inhibited colony formation more than exposure to either drug alone
(Fig. 2, A and B
, ).
|
. From the resulting lines, the x intercept
and slope were determined and then used to calculate the CI, a
parameter that indicates whether the doses of the two agents required
to produce a given degree of cytotoxicity are greater than
(CI > 1), equal to (CI = 1), or less
than (CI < 1) the doses that would be required if the
effects of the two drugs were strictly additive. When the two
treatments are assumed to be mutually exclusive, results of this
analysis are equivalent to the isobologram method (67)
.
When the effects of simultaneous treatment with CI1033 + SN-38 were analyzed under this assumption, the CI was 0.6 ± 0.1 (n = 4) at the IC50
of the combination and <1 over most of the concentrations examined
(Fig. 2D
, solid line), suggesting that the
effects were synergistic on this schedule. For completeness, the CI
calculated under the assumption that the effects of the two agents are
mutually nonexclusive is also shown in Fig. 2D
(dotted
line).
To provide an alternative assessment of the effects of combining these
two agents, T98G cells were treated with varying concentrations of
SN-38 in the absence or presence of 2 µM CI1033, a
concentration that allowed survival of >75% of the cells when used
alone. As shown in Fig. 2E
, CI1033 markedly enhanced the
effects of SN-38 at all drug concentrations.
In subsequent experiments, the effects of sequential treatment with
CI1033 and SN-38 were examined. When CI1033 was followed by SN-38, the
antiproliferative effects were slightly greater than those of either
treatment alone (Fig. 3A
; data not shown). The CI in this sequence was 1.4 ± 0.2
(n = 4) at the IC50 and 1 over
the entire range of concentrations examined (Fig. 3B)
. In
the opposite sequence, the two agents again produced greater effects
than either agent alone (Fig. 3C)
, but analysis by the
median effect method revealed that the CI was 1.2 ± 0.1
(n = 4) at the IC50 and close to
1 over the entire range of concentrations examined (Fig. 3D)
. Accordingly, the effects appeared to be synergistic
only when the two agents were administered simultaneously.
|
) and MCF-7 breast cancer cells (CI 0.12 ± 0.06 at the
IC50, n = 2), but not in PC-3
prostate cancer cells (CI = 1.1 ± 0.1 at
the IC50, n = 3; Fig. 4B
) or Ovcar-3 ovarian cancer cells (CI = 1.2 ± 0.1 at the IC50,
n = 3). When the two agents were administered
in sequence, the effects in all of these cell lines were additive or
less than additive (data not shown).
|
. In the case of
simultaneous exposure to CI1033 and CPT, the combination was less than
additive throughout the entire range of concentrations examined (Fig. 4D)
. These results indicated that the effect of combining
CI1033 with CPT derivatives varies, depending on the analogue examined.
Effect of SN-38 on EGFR Autophosphorylation.
In an initial effort to determine the basis for the schedule-dependent
synergy of CI1033 and SN-38, we evaluated potential effects of SN-38 on
EGFR tyrosine phosphorylation. As indicated in Fig. 5A
, treatment of T98G cells with CI1033 alone resulted in a rapid
inhibition of EGFR autophosphorylation. In contrast, SN-38 did not
inhibit EGFR autophosphorylation or alter EGFR polypeptide levels.
These observations argue against the possibility that SN-38 is
augmenting the effects of CI1033 by further inhibiting EGFR-mediated
signaling.
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.
During the 24 h after removal of CI1033 (the time when SN-38 would
be present in the CI1033
SN-38 sequence), there was a transient
decrease in S phase, providing a potential explanation for the
antagonism observed with this sequence. A different picture emerged
after treatment with SN-38. After a 24-h exposure to a concentration of
SN-38 that inhibited colony formation by 90%, 74% of the cells
contained >2n (S or G2-M phase) DNA content (Fig. 5B)
, indicating that the cells had been delayed in their
progression through these phases of the cell cycle. When cells were
exposed to SN-38 and CI1033 simultaneously, the cell cycle effects of
SN-38 were enhanced. At the end of the 24-h exposure, the percentage of
cells containing >2n DNA was increased to 86% (Fig. 5B)
.
Moreover, the delay at G2-M phase was prolonged: Even
48 h after drug removal, 35% of cells treated with the
combination were in G2-M phase, as compared with 21% of
cells treated with SN-38
alone.5
These results raised the possibility that CI1033 might be enhancing the
effects of SN-38.
Effects of CI1033 on SN-38 Action.
To evaluate the possible effects of CI1033 on SN-38 action in greater
detail, the effects of CI1033 on topo I polypeptide levels, topo I
activity, and topo I localization were evaluated. Treatment of T98G
cells with CI1033 for 1 or 19 h had no effect on topo I
polypeptide levels (Fig. 6A
,Lanes 1 and 7; data not shown). Likewise,
treatment of cells with CI1033 for up to 24 h had no effect on the
amount of topo I activity detected in nuclear extracts.5
Immunohistochemical studies failed to demonstrate any change in topo I
distribution within the nucleus on CI1033 treatment.5
|
100,000 as increasing amounts of topo I
became covalently bound to DNA (Fig. 6A
, Lanes
2–6). Although CI1033 alone had no effect on the topo I signal,
the addition of CI1033 resulted in a decrease in topo I signal at lower
concentrations of SN-38 (compare Lanes 2 and 8 or
Lanes 3 and 9), raising the possibility that
CI1033 was enhancing the SN-38-induced stabilization of topo I-DNA
complexes.
To evaluate this possibility in greater detail, the SN-38-induced
stabilization of protein-linked DNA single-strand breaks was evaluated
by alkaline elution. When T98G cells were treated with SN-38 for 35
min, there was a dose-dependent increase in DNA single-strand breaks
(Fig. 6B
, ). Addition of CI1033 5 min before addition of
SN-38 markedly enhanced the DNA single-strand breaks induced by any
particular concentration of SN-38 (Fig. 6B
, •). In
multiple experiments, the concentration of SN-38 required to produce a
particular level of DNA damage was decreased by a factor of
13 ± 2-fold (n = 3).
Additional studies (Fig. 6B
, inset) indicated
that CI1033 concentrations as low as 0.5 µM resulted in
enhanced ability of SN-38 to stabilize topo I-DNA complexes.
Similar studies revealed that 8 µM CI1033 also decreased
the amount of TPT required to stabilize topo I-DNA complexes by a
factor of 4 ± 0.4-fold (n = 2; Fig. 6C
). In contrast, CI1033 had almost no effect on the
ability of CPT to stabilize topo I-DNA complexes (Fig. 6D)
.
The results provide a potential explanation for the observed synergy
between CI1033 and TPT (Fig. 4C)
as well as the lack of
synergy between CI1033 and CPT (Fig. 4D)
.
CI1033 Enhances SN-38 and TPT Accumulation.
The ability of CI1033 to increase SN-38-induced topo I-DNA complexes
without altering topo I polypeptide levels raised the possibility that
CI1033 might be affecting SN-38 accumulation. To evaluate this
possibility, steady-state SN-38 levels in T98G cells were assayed by
HPLC in the absence and presence of CI1033. This analysis revealed that
SN-38 levels were 9.4 ± 1.9-fold higher in the presence
of CI1033 (Fig. 7A)
. In additional studies, TPT accumulation was also examined in the
absence or presence of CI1033. Because the excitation and emission
spectra of TPT overlap with wavelengths commonly used for flow
cytometry (25
, 28
, 68)
, we used flow microfluorometry
rather than the more laborious HPLC methodology for this analysis.
Treatment with CI1033 enhanced the steady-state accumulation of TPT by
a factor of 1.8 ± 0.2-fold in T98G cells
(n = 6; Fig. 7, B and C
). Dose-response curves revealed that this effect was
detectable at 0.25 µM CI1033 (Fig. 7B)
.
Similar analysis revealed that CI1033 also enhanced drug accumulation
in HCT8 cells but not in PC-3 cells (Fig. 7B
,
inset), providing a potential explanation for the cell line
to cell line differences in whether synergy is observed (Fig. 4, A and B)
. When the kinetics of TPT uptake and
efflux were examined in T98G cells, CI1033 had no effect on the initial
rate of TPT uptake (Fig. 7C)
but instead diminished the rate
of TPT efflux (Fig. 7D)
.
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by a factor of 7.8 ± 1.0 (n = 3). Likewise, TPT accumulation, which was decreased in MDA-MB-231/BCRP
cells compared with parental cells, was enhanced by a factor of
2.2 ± 0.5 (n = 3) in the
presence of CI1033 (Fig. 8B)
.
|
indicated that CI1033 accumulation was
4.9 ± 0.1-fold lower in cells transfected with BCRP.
This observation suggests that CI1033 is itself a substrate for BCRP,
thereby providing a potential explanation for its ability to inhibit
BCRP-mediated efflux of other agents.
Consistent with the drug accumulation results presented in Fig. 8
, as
well as the known substrate preferences of BCRP (30
, 55) ,
CI1033 sensitized the BCRP-transfected cells to the antiproliferative
effects of SN-38 (Fig. 9A)
, TPT (data not shown), and the positive control mitoxantrone (Fig. 9D)
. In contrast, CI1033 had little effect on the
antiproliferative effects of SN-38 in cells transfected with empty
vector (Fig. 9B)
. Likewise, CI1033 did not affect the action
of CPT in BCRP-transfected cells (Fig. 9C)
.
|
revealed that the BCRP polypeptide was readily detectable in HCT8 and
T98G cells. In contrast, BCRP levels were much lower in PC-3 or Ovcar-3
cells, two cell lines in which CI1033 fails to modulate SN-38
sensitivity (Fig. 4B)
.5
Additional experiments
(Fig. 10B)
indicated that the paucity of BCRP protein in
PC-3 cells reflected diminished steady-state levels of BCRP mRNA.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of the role of the HER family in a variety of neoplasms,
including adenocarcinomas of the breast, prostate, and ovary as well as
high-grade gliomas, there has been considerable interest in combining
HER family kinase inhibitors with agents that are already active in
these diseases. In anticipation of these types of clinical trials, we
evaluated the effect of combining CI1033, which is currently undergoing
Phase I clinical testing, with cisplatin, paclitaxel, or SN-38.
Clonogenic assays revealed that the combinations involving cisplatin
and paclitaxel were additive at best.5
In contrast,
combinations involving SN-38 demonstrated sequence- and cell
line-dependent synergy. In particular, the antiproliferative effects of
CI1033 and SN-38 were synergistic when T98G, HCT8, or MCF-7 cells were
treated with both agents simultaneously (Figs. 2
and 4A)
.5
When cells were treated with the two
agents sequentially, however, the results were additive at best.
Moreover, even with simultaneous treatment, CI1033 and the topo I
poisons were additive at best in PC-3 cells.
Subsequent analysis demonstrated that CI1033 concomitantly enhanced
steady-state SN-38 accumulation (Fig. 7A)
and SN-38-induced
stabilization of topo I-DNA complexes in T98G cells (Fig. 6, A and B)
. Because of the relative insensitivity
of the SN-38 assay, as well as the rapidity of drug influx and efflux,
we were unable to assess the effect of CI1033 on SN-38 uptake and
efflux. However, additional studies using flow microfluorometry also
demonstrated that CI1033 enhanced the steady-state accumulation of TPT
in T98G cells (Fig. 7, B and C)
. Subsequent
analysis using this technique revealed that CI1033 did not alter the
initial rate of TPT uptake (Fig. 7C)
but instead inhibited
drug efflux (Fig. 7D)
, providing an explanation for the
increased steady-state accumulation of TPT in the presence of CI1033.
Interestingly, drug accumulation was also enhanced in HCT8 cells (Fig. 7B
, inset), which were sensitized by CI1033, but
not in PC-3 cells, which were not sensitized by CI1033.
Additional experiments were performed to identify the transporter whose
action was modulated by CI1033. Previous studies demonstrating that TPT
and SN-38 are poor substrates for P-glycoprotein (24
, 25 , 27
, 69
, 70)
made it unlikely that inhibition of P-glycoprotein is
responsible for the dramatic sensitization observed in Fig. 2E
. Chang et al. (50)
described a
CPT-selected rodent cell line that was resistant, at least in part, as
a consequence of diminished drug accumulation. Although the transporter
involved has not been identified, our observation that CI1033 has
little or no effect on CPT-induced strand breaks (Fig. 6D)
and cytotoxicity (Fig. 4D)
made it unlikely that CI1033 was
inhibiting this transporter. More recently, several groups have
described cross-resistance between mitoxantrone and the topo I poisons
TPT and SN-38 (28
, 29 , 31)
. Subsequent studies have
identified BCRP/MXR (55
, 71) as a transporter that might
be responsible for this phenotype (30
, 31
, 68)
. In
particular, the rank order of resistance to topo I poisons in cells
that overexpress this transporter has been reported to be
SN-38 > TPT > CPT (30
, 72)
, which is the same rank order of sensitization observed with
CI1033 (e.g., Fig. 6
). These observations raised the
possibility that CI1033 might be acting as a BCRP/MXR inhibitor.
Consistent with this possibility, we demonstrated that CI1033 enhanced
the accumulation (Fig. 8)
and antiproliferative effects (Fig. 9
; data
not shown) of SN-38 and TPT in cells transfected with BCRP. In
contrast, cells transfected with empty vector were relatively
unaffected by the addition of CI1033 to SN-38 or TPT (Figs. 8
and 9)
.
Although BCRP has been previously implicated in resistance to SN-38 and
TPT (30
, 31
, 68
, 73)
, our results extend these earlier
studies in several ways. First, our analysis of BCRP-transfected cell
lines provides the first formal proof that BCRP affects the
cytotoxicity of SN-38. Previous studies performed using cells selected
in mitoxantrone or other agents demonstrated a correlation between BCRP
expression and diminished action of SN-38 but could not rule out the
possibility that a second transporter had been coselected during the
selection procedure. The recent demonstration that methotrexate
resistance in BCRP-expressing mitoxantrone-selected MCF-7 cells is
mediated by another, as yet unidentified transporter (56)
highlights the difficulty in determining drug resistance mechanisms
using only data from drug-selected cells. The present data circumvent
this problem by directly demonstrating that BCRP transfection results
in SN-38 resistance (Fig. 9, A and B)
. Second,
the present data provide the first direct evidence that BCRP is capable
of altering the accumulation of topo I poisons. Previous studies have
demonstrated diminished steady-state accumulation (30)
or
enhanced efflux (68)
of TPT in drug-selected cells, but
the present data clearly demonstrate that transfection of BCRP
diminishes accumulation of both TPT and SN-38 (Fig. 8, A and B
, respectively). Third, the present study indicates that
BCRP is endogenously expressed in certain human cancer cell lines.
Previous studies have demonstrated BCRP expression in cells that were
extensively selected for resistance to anthracyclines, mitoxantrone, or
TPT (31
, 55
, 71) . In contrast, the present results
demonstrated an effect of CI1033 on drug action in cell lines that have
not previously been exposed to these agents. Further analysis confirmed
the presence of BCRP mRNA and protein in these cell lines (Fig. 10)
,
providing evidence that a transporter capable of effluxing SN-38 and
TPT might be constitutively expressed in certain cancer cells.
The present studies also provide a potential explanation for the
ability of CI1033 to sensitize BCRP-expressing cells. The observation
that CI1033 levels are much lower in BCRP-transfected MDA-MB-231 cells
than in empty vector-transfected controls (Fig. 8C)
suggests
that CI1033 is itself effluxed by BCRP. Accordingly, CI1033 would be
expected to competitively inhibit the efflux of other agents by this
transporter. This proposed mechanism of action would account for the
fact that CI1033 must be present during the period of SN-38 exposure to
stabilize topo I-DNA complexes5
and sensitize the cells
(Figs. 2
and 3)
. We cannot, however, rule out a model in which part of
the modulatory effect of CI1033 also results from the inhibition of a
kinase whose activity regulates BCRP. Additional experiments are
required to rule out this possibility.
Additional studies are also required to determine whether the
present findings are pertinent to the modulation of clinical drug
resistance. The 10-fold decrease in SN-38 accumulation and 2-fold
decrease in TPT accumulation mediated by endogenous levels of BCRP
(Fig. 7)
are modest compared with the orders of magnitude of resistance
observed in drug-selected cells (28
, 29
, 31
, 55
, 68
, 71)
.
On the other hand, it is difficult to escalate SN-38 beyond the
currently recommended doses in the clinical setting because of severe
nonhematological toxicities. Thus, the ability of CI1033 to enhance
drug accumulation, if selective for tumor cells, might be important in
modulating clinical drug effects. Only two other compounds have been
reported to inhibit BCRP-mediated drug efflux to date, GF120918
(31
, 72 , 74)
and fumitremorgin C (73)
. In
contrast to these agents, which do not appear to have intrinsic
antineoplastic activity, CI1033 is undergoing Phase I clinical testing
as an antineoplastic agent in its own right. If its toxicity profile is
tolerable, additional preclinical and early clinical trials of
this novel sensitizing agent in combination with SN-38 appear
warranted.
|
ACKNOWLEDGMENTS |
|---|
, and Deb Strauss
for secretarial assistance. |
FOOTNOTES |
|---|
1 Primary support for this project was
provided by NIH Grants R01 CA73709 (to S. H. K.) and P31
CA15083. Additional support was provided by R01s CA88357 (to C. D. J.), CA52178 (to D. D. R.), and CA77545 (to D. D. R.) as well as grants from the Leukemia & Lymphoma Society (to
D. D. R.), Department of Veterans Affairs (to D. D. R.), the Dutch Cancer Society (NKI 99-2060), and Netherlands Asthma
Foundation (AF.35). 
2 To whom requests for reprints should be
addressed, at Division of Oncology Research, 13th Floor Guggenheim
Building, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905.
3 The abbreviations used are: topo, topoisomerase;
BCRP, breast cancer resistance protein; CI, combination index; CPT,
camptothecin; EGFR, epidermal growth factor receptor; FBS, fetal bovine
serum; HPLC, high-performance liquid chromatography; RT-PCR, reverse
transcription-PCR; SN-38, 7-ethyl-10-hydroxycamptothecin; TPT,
topotecan.
4 M. Maliepaard, G. L. Scheffer, I. F. Faneyte, M.
A. van Gastelen, A. C. L. M. Pijnenborg, A. H. Schinkel, M. J. van de
Vijver, R. J. Scheper, and J. H. M. Schellens. Subcellular localization
and distribution of the breast cancer resistance protein transporter in
normal human tissues, submitted for publication.
5 S. A. Boerner, C. Erlichman, and S. H.
Kaufmann, unpublished observations.
Received 10/11/00. Accepted 11/15/00.
|
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