Download A Synergistic Interaction between Lapatinib and Chemotherapy

Published OnlineFirst October 1, 2010; DOI: 10.1158/1535-7163.MCT-10-0197
Molecular
Cancer
Therapeutics
Preclinical Development
A Synergistic Interaction between Lapatinib and
Chemotherapy Agents in a Panel of Cell Lines Is Due
to the Inhibition of the Efflux Pump BCRP
Jackie Perry, Essam Ghazaly, Christiana Kitromilidou, Eva H. McGrowder,
Simon Joel, and Thomas Powles
Abstract
Lapatinib is a specific HER1 and 2 targeted tyrosine kinase inhibitor now widely used in combination with
chemotherapy in the clinical setting. In this work, we investigated the interactions between lapatinib and
specific chemotherapy agents (cisplatin, SN-38, topotecan) in a panel of cell lines [breast (n ¼ 2), lung (n ¼ 2),
testis (n ¼ 4)]. A high-sensitivity cell proliferation/cytotoxicity ATP assay and flow cytometry were used to
determine cell viability, apoptosis, and the effect of the drugs on cell-cycle distribution. CalcuSyn analysis was
employed to formally identify synergistic interactions between drugs. Intracellular concentrations of SN-38
were measured using a novel high-performance liquid chromatography (HPLC) technique. Flow cytometry
and HPLC techniques were used to identify the effect of lapatinib on drug influx and efflux pumps, using
specific substrates and inhibitors of these pumps. Results showed significant synergy between SN-38, and
lapatinib in the majority of cell lines (combination index < 0.75), associated with increased apoptosis. This
synergy was not universal but, when observed (Susa S/R, H1975, H358, and MDA-MB-231 cell lines), was
related to SN-38 intracellular accumulation (2.2- to 4.8-fold increase, P < 0.05 for each), attributable to the
inhibition of the breast cancer–related protein (BCRP) efflux pump by lapatinib. Flow cytometry analysis
showed that lapatinib (10 mmol/L) inhibited the efflux of mitoxantrone, a specific substrate of the BCRP
pump, in a manner similar to fumitremorgin C, a known BCRP inhibitor, confirming lapatinib as a BCRP
inhibitor. This work shows that lapatinib has a direct inhibitory effect on BCRP accounting for the synergistic
findings. The synergy is cell line dependent and related to the activity of specific efflux pumps. Mol Cancer
Ther; 9(12); 3322–9. 2010 AACR.
Introduction
Advances in molecular biology have resulted in new
targets for cancer therapy. One of the more promising of
these targets is the HER receptor family. HER1 targeted
therapies have activity in both colorectal and lung cancers, whereas HER1 and 2 targeted therapies are widely
used in breast cancer (1–3). Both monoclonal antibodies
and tyrosine kinase inhibitors (TKI) have been successfully developed in this setting. As the HER1–4 receptors
are dimerized, there is a potential advantage in targeting
Authors' Affiliation: Centre for Experimental Cancer Medicine, Institute of
Cancer, Barts and the London School of Medicine, Queen Mary College,
London, UK
Note: Supplementary material for this article is available at Molecular
Cancer Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Simon Joel, Centre for Experimental Cancer
Medicine, Institute of Cancer, John Vane Science Centre, Barts and the
London School of Medicine, Queen Mary College, Charterhouse Square,
London EC1M 6BQ, UK. Phone: 44-207-8823821; Fax: 44-207-8823891.
E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-10-0197
2010 American Association for Cancer Research.
3322
more than 1 of these receptors concurrently (4). Lapatinib,
a 4-anilinoquinonazoline, is one such agent that targets
both HER1 and 2 receptors. Lapatinib has proven efficacy
in both preclinical and clinical models (3, 5, 6). It has
impressive single-agent activity and is being investigated
in a range of malignancies (7). However, the most promising clinical data are seen when it is used in combination with chemotherapy, although the reasons for this are
unclear. Preclinical data show that lapatinib downregulates nucleotide synthesis-related genes such as thymidylate synthase and that it may result in synergistic
interactions with other chemotherapy agents (8). At the
same time, it is apparent that HER targeted TKIs may
affect drug efflux pumps (9, 10).
Drug efflux pumps are expressed in a broad spectrum
of tissues, including the plasma membrane in stem cells,
placenta, liver, small intestine, colon, lung, and kidney,
suggesting their role in the protection/detoxification of
xenobiotics (11, 12). They are also expressed in cancer
tissues and are important in determining resistance to
specific chemotherapy agents such as the taxanes and
topoisomerase inhibitors (13). Drug efflux is largely
mediated by the ATP-binding cassette (ABC) transporters
p-glycoprotein (P-gp) ABCB1 and breast cancer–related
Mol Cancer Ther; 9(12) December 2010
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Published OnlineFirst October 1, 2010; DOI: 10.1158/1535-7163.MCT-10-0197
A Synergistic Interaction between Lapatinib and Chemotherapy
Table 1. EC50 values for drug compounds studied in breast, lung, and TGCT cell lines (fitted values from
at least 3separate experiments with 95% confidence interval)
Cell line
EC50 (95% confidence interval) values (cell viability)
Lapatinib, mmol/L
SN-38, nmol/L
Cisplatin, mmol/L
Topotecan, nmol/L
Paclitaxel, nmol/L
7.0 (6.2–7.9)
6.4 (3.3–12.3)
8.7 (5.4–13.9)
20 (12.6–31.7)
4.0 (3.7–4.4)
3.8 (2.9–4.8)
20 (10–40)
30 (20–30)
40 (10–100)
170 (90–340)
9.8 (5.7–16.9)
7.2 (5.5–9.5)
N/A
N/A
13.5 (9.8–18.5)
13.9 (11.0–17.6)
0.50 (0.46–0.56)
1.9 (1.6–2.3)
20 (9–40)
30 (20–60)
1,240 (1,150–1,340)
1,420 (1,040–1,920)
N/A
N/A
0.013 (0.012–0.014)
N/A
20 (10–30)
14 (10–19)
5.1 (4.3–6.1)
4.7 (3.8–5.8)
MCF-7
MDA-MB-231
H358
H1975
Susa S
Susa R
NOTE: Breast, lung, and TGCT cell lines were treated at 0 and 5 nonzero concentrations of each study drug for 72 h. Viability was then
determined using a high-sensitivity cell proliferation/cytotoxicity kit (Vialight HS, Lonza). EC50 values with 95% confidence interval
were then determined by Graphpad Prism, Version 3.03. Results presented are from a minimum of 3 separate experiments.
protein (BCRP) ABCG2. Although these efflux pumps can
translocate unique compounds, there is an overlap with
some drug substrates. Drugs transported by ABCB1
include hydrophobic compounds such as Vinca alkaloids,
anthracyclines, taxanes, and epidophyllotoxins. The spectrum of drugs transported by ABCG2 includes mitoxantrone (MXR), anthracyclines, topoisomerase I inhibitors,
methotrexate, and flavopiridol. The situation is further
complicated by the potential role of influx pumps in this
setting, which results in a balance between influx and
efflux. Relatively little is known about the effects of the
TKIs on these efflux and influx pumps. Initial reports
suggest that specific agents such as gefitinib and lapatinib
may inhibit P-gp or BCRP (14, 15).
In the work presented here, we initially investigated
potential interactions between lapatinib and a spectrum
of chemotherapy agents, some of which are ABC transporter substrates. This was followed by experiments to
specifically address the mechanism underlying the interactions observed, with a focus on the BCRP efflux pump.
Initially, we used high-performance liquid chromatography (HPLC) to determine the effects of drugs on intracellular concentration of SN-38 and then used HPLC and
flow cytometry to investigate the effects of drugs with
substrates as well as inhibitors of these pumps.
SN-38 is the active metabolite of irinotecan (a topoisomerase I inhibitor). This family of drugs (irinotecan and
topotecan) is widely used in the treatment of lung and
ovarian cancer. SN-38 was chosen as the focus of the
efflux pump experiments because a synergistic interaction was observed between SN-38 and lapatinib in our
original combination experiments.
Material and Methods
Cell lines
The Susa cell line pair was obtained from University
College Hospital (Prof. Masters, London, UK). All other
cell lines were sourced from the American Type Tissue
www.aacrjournals.org
Collection. MCF-7 and MDA-MB-231 (breast) cancer cells
were cultured in DMEM media. H1975, H358 (lung), Susa
S (TGCT) and its cisplatin-resistant pair were all cultured
in RMPI 1640. All media were supplemented with 10%
FCS and 1% Pen/strep, and all cultures were maintained
at 37 C, 5% CO2. Although the H358 cell line has only 1
mutation in EGFR (L858R), H1975 cells harbor both
L858R and T790M EGFR mutations. The T790M substitution in EGFR was found to confer resistance to TKIs
including lapatinib (16, 17). An overview of the cell lines
can be found in Supplementary Data, Table 1. Cell lines
were authenticated using DNA profile (short tandem
repeat, STR) analysis and tested for mycoplasma
(MycoAlert mycoplama detection kit, Lonza) on a regular
basis (this most recently occurred in May 2010).
Cytotoxicity assay
During exponential growth phase, cells were trypsinized and harvested for single-agent and combination
drug activity experiments. Cells were then seeded into
flat-bottomed 96-well plates at a concentration of 5 104
cells/mL and placed in an incubator at 37 C, 5% CO2,
overnight to adhere. Study drugs were then added to the
plates in triplicate at 0 and 5 nonzero concentrations.
Cells were cultured for a further 72 hours, after which
drug effects were determined using a high-sensitivity cell
proliferation/cytotoxicity assay based on the determination of intracellular ATP (Vialight HS kit, Lonza). Plates
were read on a Polarstar Optima plate reader (BMG
Labtech).
The EC50 values of each agent were determined using a
sigmoidal EMAX method in Prism 5 (Graphpad Software)
from which concentrations were established for use in
combination experiments. Drugs were given simultaneously for 72 hours for the combination experiments.
Combination data was then analyzed using CalcuSyn
software (Version 2; Biosoft). Combinations with a combination index (CI) < 1 were considered synergistic and
with CI < 0.5 highly synergistic.
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Perry et al.
Cell-cycle analysis by flow cytometry
Cells were seeded at 1 105 cells/mL in 6-well plates
and allowed to adhere overnight. Single agents or drug
combinations were then added directly to the culture
media in these wells and incubated for a further 72 hours.
Cells were then pelleted by centrifugation at 200 g for 6
minutes after trypsinization, washed in ice-cold Hanks
buffer, fixed using 70% ethanol, and stored at 30 C. On
the day of analysis, cells were pelleted by centrifugation
(200 g for 6 minutes), washed in ice-cold Hanks buffer to
remove the ethanol, resuspended in PI (50 mg/mL)/
RNase (10 mg/mL) solution, and kept on ice in the dark
until analysis. Samples were run on a FACSCalibur flow
cytometer (Becton Dickinson), using CellQuest software,
Version 3.3. The resulting data were then analyzed using
winMDI, Version 2.8.
BCRP activity by flow cytometry
Cells were seeded at 1 105 cells/mL in 12-well
plates and allowed to grow for 72 hours, after which
the media was removed and replaced with 1 mL of fresh
media for uptake and efflux assays. Mitoxantrone, 10
mmol/L, was used as a specific substrate for measuring
BCRP activity. Cyclosporine A (CA), 10 mmol/L, and
fumitremorgin C (FTC), 5 mmol/L, were used as known
inhibitors of P-gp and BCRP, respectively, when investigating the effect of lapatinib, 10 mmol/L, on uptake
and efflux. Intracellular MXR was determined using the
FL-3 (670 nm) detector on a FACSCalibur flow cytometer. As the interactions seen were universal in all
the cell lines, only representative cell lines with clinical
relevance are presented. Experiments investigating the
effect of MXR with both lapatinib and SN-38 together
were not performed, as SN-38 is also a BCRP substrate
and would result in both SN-38 and lapatinib competing
with MXR for BCRP efflux.
Uptake Assays. Drugs/inhibitors were added
directly to the fresh media in each well and incubated
at 37 C for 2 hours to allow their uptake. Cells were then
harvested by trypsinization, washed rapidly in ice-cold
Hanks buffer, and placed on ice until analysis.
Efflux Assays. After drug/inhibitor uptake, positive
control samples (no efflux period) were trypsinized,
washed in ice-cold Hanks buffer, and placed on ice prior
to the immediate determination of MXR. For other treatments, cells were washed in ice-cold Hanks buffer and
fresh media containing the various inhibitors to be investigated was then added. Samples were incubated for a
further 2 hours to allow efflux to occur. After the efflux
period, cells were harvested by trypsinization, washed in
ice-cold Hanks buffer, and placed on ice for immediate
analysis by flow cytometry.
Intracellular SN-38 accumulation
Intracellular SN-38 concentration was measured
using a novel HPLC assay. Cells were trypsinized
and reconstituted in cell culture media at a concentration of 2 106/mL. Drugs were then added to 3 mL of
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the reconstituted cells at the following concentrations:
SN-38 (0.1 mmol/L), lapatinib (10 mmol/L), disodium
4,40 -diisothiocyanatostilbene-2,20 -disulfonate
(DIDS,
organic anion transport inhibitor, 100 mmol/L), CA
(10 mmol/L), and FTC (5 mmol/L). Cells were incubated
at 37 C, 5% CO2, for 2 hours, washed twice with ice-cold
Hanks buffer, and reconstituted in 1 mL of ice-cold
Hanks buffer. After centrifugation (900 g, 4 C for 5
minutes), the supernatant was removed and 300 mL of
cell extraction solution [ice-cold methanol containing
0.1 mol/L of HCl and 2 mmol/L of the internal standard
camptothecin (CPT)] was added to the cell pellet. The
extraction solution and cell pellet were vortex mixed for
1 minute, kept on ice for a further 10 minutes, and then
centrifuged at 20,000 g at 4 C for 10 minutes. Two
hundred fifty microliters of the resulting supernatant
was removed and evaporated to dryness under a gentle
air stream. Dried extracts were reconstituted in 250 mL
of 50-mmol/L NaH2PO4 (pH ¼ 3.1). The solution was
vortex mixed and transferred to HPLC vials. Separation
of SN-38 and CPT was carried out using a Luna ODS
column (3 mmol/L, 150 4.6-mm ID; Phenomenex) and
an isocratic mobile phase (50 mmol/L of NaH2PO4, pH
¼ 3.1, containing 10 mmol/L of sodium dodecyl sulfate
65%:acetonitrile 35%) at a flow rate of 1 mL/min with
fluoresence detection (excitation, 380 nm; emission, 540
nm). As the interactions seen were universal in all the
cell lines, representative cell lines with clinical relevance
are presented in the article.
Results
Activity of lapatinib alone and in combination
with chemotherapy drugs
The activity of lapatinib and the cytotoxic drugs studied was summarized to an EC50 value, as shown in
Table 1. These values varied markedly and were cell line
and drug dependent. Of particular note was the lack of
cross-resistance to lapatinib in the TGCT cell line with
cisplatin-sensitive and -resistant sublines.
The effect of the chemotherapy drugs in combination
with lapatinib was then investigated. CalcuSyn plots and
CI values for some of the combinations are presented in
Tables 2 and 3 (and Supplementary Data A). Clear
synergy was noted between SN-38 and lapatinib in
the majority of cell lines (CI < 0.75), with strong synergy
(CI < 0.5) in most (Table 2). The exception to this was
MCF-7 cells in which the effects were additive (Table 2,
Supplementary Data Fig. A) or antagonistic. No clear or
consistent synergistic interaction was seen between cisplatin or paclitaxel and lapatinib (Supplementary Data
and Table 2).
To investigate whether the interaction with SN-38 and
lapatinib was a class effect, the experiments were
repeated with topotecan (also a topoisomerase I inhibitor). Marked synergy was also identified (CI < 0.5 at most
concentrations; Supplementary Data Fig. A). Therefore,
the interaction was a class effect.
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A Synergistic Interaction between Lapatinib and Chemotherapy
Table 2. Combination index values for the combination of lapatinib with SN-38 and cisplatin (CDDP) in a
panel of cell lines
Lapatinib, mmol/L, with SN-38, mmol/L
Susa S
Lap/SN-38
0.5/0.001
1/0.005
2/0.01
4/0.05
8/0.1
Susa R
H1975
H358
MDA-MB-231
MCF-7
CI
Lap/SN-38
CI
Lap/SN-38
CI
Lap/SN-38
CI
Lap/SN-38
CI
Lap/SN-38
CI
0.43
0.51
0.54
0.42
0.15
0.5/0.001
1/0.005
2/0.01
4/0.05
8/0.1
0.39
0.26
0.31
0.30
0.15
1/0.01
3/0.05
10/0.1
15/0.5
30/1.0
0.11
0.19
0.28
0.75
0.85
0.1/0.001
0.5/0.005
1/0.01
3/0.05
10/0.1
0.20
0.38
0.32
0.67
0.96
0.5/0.001
1/0.01
2/0.02
4/0.03
8/0.05
1.296
0.48
0.18
0.16
0.32
2.5/0.001
5/0.005
7.5/0.01
10/0.05
20/0.1
2.95
1.08
1.53
2.03
3.58
Lapatinib, mmol/L with cisplatin (CDDP), mmol/L
Susa S
Lap/CDDP
0.5/0.3
1/1
2/3
4/10
8/30
Susa R
H1975
H358
CI
Lap/CDDP
CI
Lap/CDDP
CI
Lap/CDDP
CI
0.691
0.968
1.509
1.707
1.187
0.5/1
1/3
2/10
4/30
8/100
0.759
1.069
1.391
0.627
0.149
1/1.0
3/3
10/5
15/10
30/20
99.379
298.14
0.89
0.652
0.508
0.1/1
0.5/3
1/5
3/10
10/20
0.598
0.631
0.592
0.923
1.48
NOTE: CI values of < 0.5 are shown in bold. The CalcuSyn analysis included data from 4 separate experiments.
Abbreviations: CI, combination index; Lap, lapatinib.
Effect of lapatinib alone or in combination
on cell-cycle distribution
Flow cytometry data for the individual chemotherapy
agents showed a concentration-dependent increase in
apoptosis, with a G2/M-phase-arrested population with
SN-38. Lapatinib had little or no effect on cell-cycle
Table 3. Effect of lapatinib (10 mmol/L) on the
accumulation of SN-38 in a panel of cell lines as
measured by a novel HPLC method
Cell line
Intracellular SN-38
concentration, nmol/L
SN-38 alone
Susa S
Susa R
H1975
H358
MDA-MB-231
MCF-7
5.7 0.7
4.8 0.4
7.2 1.9
9.1 2.3
9.2 4.0
21.2 1.3
SN-38 þ Lap
14.6
11.0
34.4
23.8
18.5
19.2
1.1
0.6
7.2
5.9
7.1
1.2
NOTE: Breast, lung, and TGCT cell lines were preloaded
with SN-38 (0.1 mmol/L) for 2 h, at 37 C, 5% CO2, in the
presence or absence of lapatinib (10 mmol/L). Data shown
are the mean SD of 3 separate experiments.
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distribution, apart from a small increase in S-phase cells
in some cell lines.
When given in combination, the synergistic combinations were associated with a marked increased in apoptosis
(P < 0.05; Supplementary Data Fig. B). In MCF-7 cells, the
combination of lapatinib and SN-38, which was antagonistic in cell viability assays, resulted in a significant
decrease in the apoptotic fraction with an increased G2/
M-phase-arrested population (Supplementary Data Fig. B).
Effect of lapatinib on the intracellular accumulation
of SN-38
Cells that were pretreated with lapatinib showed a
significant increase in intracellular SN-38 concentration
compared with cells with SN-38 alone (Table 3, 2.2- to 4.8fold increase, P < 0.05 for each; and Supplementary
Fig. C). The exception was the MCF-7 cell line, in which
no significant change was observed (Table 3). As the
MCF-7 cell line did not exhibit synergy between SN-38
and lapatinib, increased SN-38 accumulation in the presence of lapatinib is a potential mechanism underlying
the synergistic interactions observed.
The effect of drug uptake and efflux inhibitors on
intracellular SN-38 concentration was further investigated in the H1975 and H358 cell line pair. Lapatinib
(10 mmol/L) resulted in a 4.8- and 2.6-fold increase in SN38 in H1975 and H358 cells, respectively. The influx
inhibitor DIDS (100 mmol/L), which inhibits multispecific
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Table 4. Effect of lapatinib and influx/efflux pump inhibitors on the accumulation of SN-38 in H1975 and
H358 cell lines
Drug treatments
SN-38
SN-38
SN-38
SN-38
SN-38
SN-38
SN-38
(0.1 mmol/L)
þ lapatinib (10 mmol/L)
þ DIDS (100 mmol/L)
þ lapatinib (10 mmol/L) plus; DIDS (100 mmol/L)
þ CA (10 mmol/L)
þ FTC (5 mmol/L)
þ CA þ FTC
SN-38 concentration
in H1975 cells, nmol/L
7.2
34.4
5.0
26.6
14.2
31.2
35.9
1.9
7.2
0.4
1.9
3.7
7.3
8.3
SN-38 concentration
in H358 cells, nmol/L
9.1
23.8
6.0
14.8
12.3
24.6
23.2
2.3
5.9
1.3
0.9
3.6
4.2
7.2
NOTE: Cells were preloaded with SN-38 (0.1 mmol/L) for 2 h, at 37 C, 5% CO2, in the presence or absence of lapatinib (10 mmol/L) or
various other known uptake/efflux pump inhibitors. SN-38 concentration was then measured by HPLC analysis. Data shown are the
mean SD from 3 separate experiments.
Abbreviations: CA, cyclosporine A; DIDS, disodium 4,40 -diisothiocyanatostilbene-2,20 -disulfonate; FTC, fumitremorgin C.
organic anion transport, inhibited the influx of SN-38 by
36%. DIDS had a similar effect on SN-38 concentration in
the presence of lapatinib, suggesting that lapatinib did
not have a significant effect on influx pumps (P > 0.05).
(Table 4). FTC, an established BRCP inhibitor, had an
effect on SN-38 accumulation similar to that seen with
lapatinib (4.3- and 2.7-fold accumulation of SN-38 in
H1975 and H358 cells, respectively, compared with 4.8and 2.6-fold increase with lapatinib), suggesting lapatinib
may result in SN-38, and other, drug accumulation via
BCRP inhibition.
Cyclosporine A (CA, 10 mmol/L), a P-gp inhibitor, had
a significant effect on SN-38 concentration in only one of
the cell lines (H1975). However, the addition of CA to
FTC had no increased effect compared with FTC alone
(P > 0.05; Table 4), suggesting that SN-38 accumulation
with lapatinib is due to BCRP inhibition in these cell lines.
Analysis of the effects of lapatinib on BCRP function
by flow cytometry
After establishing that lapatinib resulted in the accumulation of SN-38, most likely by inhibiting BCRP-mediated
drug efflux, we used flow cytometry in 3 cell lines (H1975,
H358, and MDA-MB-231) to confirm these findings, using
the fluorescent BCRP substrate MXR.
Flow cytometry data showed a reduction in the fluorescent MXR signal after 2 hours of incubation at 37 C,
representing MXR efflux (Figs. 1 and 2). Lapatinib inhibited this MXR efflux to the same extent as FTC, confirming lapatinib as a BCRP inhibitor. The effect of CA (an
inhibitor of P-gp) was typically between that of no inhibitor (efflux) and lapatinib or FTC, in all 3 lines. This
supported the HPLC data that showed only a modest
effect of CA (Table 4).
Pazopanib (a VEGF TKI) and SN-38
To establish whether the synergistic interactions seen
with lapatinib and SN-38 were not exclusive to HER
targeted TKIs, we investigated whether the same effects
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Mol Cancer Ther; 9(12) December 2010
were seen with pazopanib (a VEGF TKI). These were
investigated in 2 paired testis cancer cell lines (Susa S and
Susa R). No synergistic interactions were observed (CI > 1
for each).
Discussion
TKIs are increasingly being used in combination with
chemotherapy agents with the aim of improving the
survival of cancer patients. Lapatinib is a prominent
HER1 and HER2 TKI (Fig. 3) that has been successfully
used with a spectrum of agents (3). The in vitro work
presented here identified a synergistic interaction
between SN-38 and lapatinib. This interaction was not
Figure 1. Flow cytometry data showing the effect of lapatinib (Lap),
cyclosporine A (CA), and fumitremorgin C (FTC) on mitoxantrone (MXR)
efflux through BCRP. Breast and lung cancer cell lines were preloaded
with MXR (a specific substrate of BCRP) for 30 minutes at 37 C, 5% CO2,
after which efflux was allowed to occur in the presence or absence of
lapatinib (10 mmol/L), CA (10 mmol/L, a specific P-gp inhibitor), or FTC
(5 mmol/L, a specific BCRP inhibitor). Data shown are the mean SD of a
minimum of 3 separate experiments, *P < 0.05 compared with control
(uptake).
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A Synergistic Interaction between Lapatinib and Chemotherapy
exclusive to SN-38, occurring with another topoisomerase I inhibitor (topotecan), suggesting a class effect. The
synergy was seen across a range of concentrations. Cellcycle data showed that these effects were associated with
A
H358
80
FTC 5 µmol
Lap 10 µmol
Efflux
-ve Control
Figure 3. Chemical structure of lapatinib.
0
Events
MXR uptake
100
101
B
102
103
H1975
64
FTC 5 µmol
MXR uptake
Efflux
-ve Control
0
Events
Lap 10 µmol
100
101
C
102
103
122
MDA-MB-231
FTC 5 µmol
Efflux
Lap 10 µmol
Events
-ve Control
0
MXR uptake
101
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102
103
an increased sub-G1 (apoptotic) population. However,
these synergistic interactions were not universal in all
cell lines, with a lack of synergy in the MCF-7 cell line. In
addition, the interaction was TKI specific, in that a
synergistic interaction was not seen with pazopanib (a
VEGF TKI) and SN-38.
In view of previous reports suggesting an effect of
lapatinib on drug efflux pumps, particularly BCRP
(14, 18), we developed a novel HPLC method to determine
the effect of lapatinib on intracellular SN-38 concentrations. These results confirmed an increase in intracellular
SN-38 in the presence of lapatinib, further confirmed by a
lapatinib-mediated increase in MXR by using flow cytometry. This suggests that inhibition of the BCRP efflux
pump by lapatinib was responsible for the synergistic
interaction in the H358 and H1975 lung cancer cell lines
and in the breast cancer cell line MDA-MB-231. BCRP has
previously been reported to be involved in the efflux of
topoisomerase I inhibitors (19), but the intracellular SN-38
concentration data presented here are the strongest evidence to date for this direct pharmacokinetic effect of
lapatinib and have not been described previously.
This synergistic interaction was not observed in all
cell lines studied. Indeed, no synergy was seen between
SN-38 and lapatinib in the MCF-7 cell line. This cell line
overexpresses P-gp rather than BCRP, which is less
important for the efflux of topisomerase I inhibitors
(14, 20–22). The relative expression and activity of different ABC efflux transporters may, therefore, be important
in determining the degree of synergistic interaction
between cytotoxic agents and lapatinib and potentially
between TKIs (10, 23). Our experiments measured pump
activity rather than protein level, which is theoretically
advantageous in terms of understanding the mechanism
of the interactions seen. However, the mechanism by
which lapatinib inhibits BCRP remains unknown and
requires further investigation.
Figure 2. Representative flow cytometry histograms illustrating the effect
of lapatinib (Lap) and fumitremorgin C (FTC) on mitoxantrone (MXR) efflux
through BCRP. Lung (A and B) and breast cancer (C) cell lines were
preloaded with MXR (a specific substrate of BCRP) for 30 minutes at 37 C,
5% CO2 (MXR uptake), after which efflux was allowed to occur in control
cells (efflux), or the presence or absence of lapatinib (10 mmol/L) or FTC (5
mmol/L, a specific inhibitor of BCRP). In the left-most peak in each panel,
"-ve Control" refers to negative control.
Mol Cancer Ther; 9(12) December 2010
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3327
Published OnlineFirst October 1, 2010; DOI: 10.1158/1535-7163.MCT-10-0197
Perry et al.
Efflux pumps are expressed in nonmalignant tissue
such as the kidneys as well as in tumors. Therefore, these
results have implication in terms of drug toxicity as well
as efficacy (24).
Other groups have investigated potential interactions
between lapatinib and cytotoxic chemotherapy agents.
Coley and colleagues demonstrated that cisplatin and
lapatinib were synergic in an ovarian cancer cell line
(9). This is in contrast to our data, which show no synergistic interaction. Indeed, cisplatin is not excreted from the
cells by either P-gp or BCRP, which is why cisplatin was
chosen as a negative control for our experiments. Cisplatin
is not associated with excretion via an efflux pump, and no
specific mechanism was identified in this work. Moreover,
McHugh and colleagues also investigated the combination of cisplatin and lapatinib with a number of sequencing experiments, demonstrating antagonism when the
drugs were given together but some evidence of synergy
with certain sequences of exposure to each agent (25). The
reasons for this are unclear and require further investigation. However, this emphasizes the time-dependent nature of potential interactions between lapatinib and other
agents and suggests that other mechanisms may be implicated that are independent of drug efflux pumps.
The concentrations of lapatinib required to achieve
these synergistic interactions are clinically relevant
(10). This is evident from phase I clinical data investigating lapatinib and irinotecan (SN-38 is the active metabolite of irinotecan) in combination, which showed that
lapatinib significantly altered the pharmacokinetics of
SN-38, with an increase in the area under the plasma
concentration–time curve of SN-38 for the combination
(26). The data presented here therefore have clinical
implications, particularly as drug combinations are being
given in large randomized studies, whereas unacceptable
toxicity has been noted with other combinations (27, 28).
The data presented here are somewhat dependent on
the specificity of established substrates and inhibitors of
efflux pumps. Although the optimal agents available
were used, it is conceivable that cross activity with other
efflux pumps occurs. This was particularly true in the 2
lung cancer cell lines for which the experiments suggested that MXR had some efflux through P-gp or that
CA shows some cross-selectivity for both P-gp and BCRP.
The effect of TKIs on influx pumps is established in
hematologic malignancies (29). Our work showed that
lapatinib did not increase the influx of SN-38 via organic
anion transporters, as coincubation with DIDS had little,
if any, effect on SN-38 concentration.
SN-38 is the active metabolite of irinotecan, which is
widely used in cancer medicine. This interaction with
lapatinib has clinical implications in tumor types, such as
lung cancer, where both HER1 and HER2 targeted therapy
and topoisomerase I inhibitors are given concurrently.
In summary, lapatinib has an inhibitory effect on BCRP
resulting in a synergistic interaction with topoisomerase I
inhibitors mediated by increased intracellular drug concentration. This effect is cell-line dependent and results in
specific synergistic interactions. These data have clinical
significance in the further development of lapatinib combinations.
Disclosure of Potential Conflicts of Interest
D. Powles received an educational grant from GlaxoSmithKline.
Grant Support
This work was financially supported by the Orchid Cancer Appeal and the
Barry Reed Research Fund.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received 03/19/2010; revised 09/22/2010; accepted 09/27/2010;
published OnlineFirst 10/01/2010.
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A Synergistic Interaction between Lapatinib and Chemotherapy
Agents in a Panel of Cell Lines Is Due to the Inhibition of the Efflux
Pump BCRP
Jackie Perry, Essam Ghazaly, Christiana Kitromilidou, et al.
Mol Cancer Ther 2010;9:3322-3329. Published OnlineFirst October 1, 2010.
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