Download use of the proton pump inhibitor pantoprazole to modify the

USE OF THE PROTON PUMP INHIBITOR PANTOPRAZOLE TO MODIFY THE
DISTRIBUTION AND ACTIVITY OF DOXORUBICIN: A POTENTIAL STRATEGY TO
IMPROVE THE THERAPY OF SOLID TUMORS
Krupa J. Patel1, Carol Lee1, Qian Tan1 and Ian F. Tannock1,2
1
Department of Medical Biophysics, and 2 Division of Medical Oncology and Hematology,
Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada
Corresponding Author: Ian F. Tannock: [email protected]
Krupa J. Patel: [email protected]
Qian Tan: [email protected]
Carol Lee: [email protected]
Address for correspondence:
Ian F. Tannock MD, PhD, DSc
Princess Margaret Hospital and University of Toronto,
610 University Avenue, Suite 5-208,
Toronto ON M5G 2M9, Canada
Tel 416 946 2245
Fax 416 946 4563
1
Statement of Translational Relevance
The tumor microenvironment is likely to play an important role in drug resistance. Our
group and others have shown that limited drug distribution may influence the therapeutic
efficacy of anti-cancer drugs. This article examines the use of the proton pump inhibitor
pantoprazole in combination with doxorubicin in in vitro and in vivo model systems.
Pantoprazole was found to increase doxorubicin uptake and penetration through tissue in in vitro
studies. In solid tumors, pantoprazole was found to improve doxorubicin distribution and
combined treatment increased tumor growth delay in mice. This study suggests that
pantoprazole may improve the therapeutic efficacy of doxorubicin by altering intracellular and
extracellular drug distribution.
2
Abstract
Introduction: Limited drug distribution within solid tumors is an important cause of drug
resistance. Basic drugs (e.g. doxorubicin) may be sequestered in acidic organelles thereby
limiting drug distribution to distal cells and diverting drugs from their target DNA. Here we
investigate the effects of pantoprazole, a proton pump inhibitor, on doxorubicin uptake, and
doxorubicin distribution and activity using in vitro and murine models. Methods: Murine
EMT-6 and human MCF-7 cells were treated with pantoprazole to evaluate changes is
endosomal pH using fluorescence spectroscopy, and uptake of doxorubicin using flow
cytometry. Effects of pantoprazole on tissue penetration of doxorubicin were evaluated in
multilayered cell cultures (MCCs), and in solid tumors using immunohistochemistry. Effects of
pantoprazole to influence tumor growth delay and toxicity due to doxorubicin were evaluated in
mice. Results: Pantoprazole (>200μM) increased endosomal pH in cells, and also increased
nuclear uptake of doxorubicin. Pretreatment with pantoprazole increased tissue penetration of
doxorubicin in MCCs. Pantoprazole improved doxorubicin distribution from blood vessels in
solid tumors. Pantoprazole given prior to doxorubicin led to increased growth delay when given
as single or multiple doses to mice bearing MCF7 xenografts. Conclusions: Use of
pantoprazole to enhance the distribution and cytotoxicity of anticancer drugs in solid tumors
might be a novel treatment strategy to improve their therapeutic index.
Keywords
Drug distribution, proton-pump inhibitors, tumor microenvironment, doxorubicin
3
Introduction
Drug resistance limits treatment of cancer by chemotherapy. For a tumor to respond to
chemotherapy a drug must leave tumor blood vessels efficiently and distribute throughout tumor
tissue to reach all cancer cells in concentrations that will lead to cytotoxicity (1, 2). The
distribution of anticancer drugs such as doxorubicin is limited in solid tumors and this limited
distribution may be an important mechanism of drug resistance (3-9).
Many solid tumors develop regions of extracellular acidity due to production and poor
clearance of carbonic and lactic acid (10-13). The pH gradient between an acidic extracellular
environment and a neutral-alkaline intracellular pH may influence drug uptake and activity.
Also, cells contain acidic organelles such as lysosomes and endosomes (14-16). Since many
chemotherapeutic drugs such as anthracyclines and vinca alkaloids are weak lipophilic bases,
they readily enter acidic organelles where they are protonated and sequestered (17).
Vacuolar-H+-ATPases (V-H+-ATPases) transport H+ ions across the membranes of a wide
array of intracellular compartments and are the major mechanism for regulation of endosomal
pH (18). Agents that disrupt the pH gradient between the cytoplasm and endosomes in tumors
might decrease the sequestration of basic anticancer drugs and render cells more sensitive to the
drug. A class of H+-ATPase inhibitors called proton pump inhibitors (PPIs) inhibit acidification
of cells in the wall of the stomach and are used clinically for treating patients with peptic ulcer
disease. These agents also inhibit V-H+-ATPase albeit at somewhat higher concentrations then is
required to inhibit acidification in the stomach (19). PPIs accumulate selectively in acidic spaces
and with the inhibition of V-H+-ATPase activity, increase both extracellular pH and the pH of
acidic organelles (20). Pretreatment with a PPI might alter intracellular drug distribution by
inhibiting drug sequestration, thereby allowing more drug to enter the nucleus and cause
4
cytotoxicity, and to exit the cell and be taken up by cells distant from blood vessels. We
hypothesized that preventing sequestration of drug in acidic endosomes would improve
extracellular drug distribution. In support of this hypothesis modest improvements in drug
distribution have been observed in multilayered cell cultures (MCCs) pretreated with
chloroquine or the PPI, omeprazole (21). As well, proton pump inhibitors have been shown to
be increase chemotherapy efficacy in vitro and improve tumor control in vivo (22-24).
Here we describe and evaluate the role of pantoprazole, which has been reported (or
might be expected) to increase endosomal pH, for its effects on uptake of doxorubicin into tumor
cells, and its distribution within them. We then describe experiments to characterize the effects
of pantoprazole to modify the distribution and activity of doxorubicin in solid tumors.
Materials and Methods
Drugs and reagents
Doxorubicin (Pharmacia, Mississauga, Canada) was purchased from the hospital
pharmacy as a solution at a concentration of 2 mg/mL. Purified rat anti-mouse CD31
(platelet/endothelial adhesion molecule 1) monoclonal antibody was purchased from BD
PharmMingen (Mississauga, Canada), and Cy3-conjugated goat anti-rat IgG secondary antibody
was purchased from Jackson ImmunoResearch Laboratories, Inc. (Pennsylvania, USA).
Pantoprazole (Nycomed, Oakville, Canada) was purchased from the hospital pharmacy as a
lyophilized powder and dissolved in 0.9% saline. Radiolabeled doxorubicin was obtained from
Amersham Life Sciences (Buckinghamshire, United Kingdom). Lansoprazole was purchased
from Sigma (St. Louis, MO) and dissolved in ethanol.
5
Cell lines
The parental mouse mammary sarcoma EMT-6 was provided originally by Dr Peter
Twentyman, Cambridge UK. The human breast cancer cell line MCF-7 and the human vulvar
epidermoid carcinoma cell line A431 were obtained from the American Type Culture Collection
(ATCC; Virginia, USA).
EMT-6 and MCF-7 cells were maintained as monolayers in α-MEM media,
supplemented with 10% fetal bovine serum, (FBS; Hyclone, Logan, UT). A431 cells were
maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS. All media
were obtained from the hospital media facility. Cells were grown in a humidified atmosphere of
95% air and 5% CO2 at 37˚C. Routine tests to exclude mycoplasma were performed.
Tumors were generated by subcutaneous injection of 1 – 5 x 106 exponentially growing
cells into the left and right flank regions of female athymic nude mice (MCF7 and A431) or
syngeneic Balb/C mice (EMT-6). Estrogen pellets were implanted into the nude mice one day
prior to injection of MCF-7 tumor cells. All procedures were carried out following approval of
the Institutional Animal Care Committee.
Measurement of Endosomal pH
To measure endosomal pH, EMT6 cells (106/ml) were treated with varying
concentrations of lansoprazole, pantoprazole, or tamoxifen (included because of prior reports of
effects to raise endosomal pH, (25)), and MCF7 cells were treated with varying concentration of
pantoprazole in the presence or absence of doxorubicin (1.8µM). They were incubated for 3
hours with dextran-fluorescein-tetramethylrhodamine 10,000 MW, anionic (FITC/TMR-dextran,
Molecular Probes, Inc. Eugene, OR) which is taken up into endosomes, followed by exposure to
media for 2 hours. Fluorescence was measured using a Coulter Epics Elite flow cytometer
6
(Beckman Coulter, Miami, FL) equipped with an argon laser emitting at 488nm. The argon laser
was used to excite FITC and TMR with emission evaluated at 525nm (pH-dependent) and
575nm (pH-independent) respectively. Calibration of fluorescence measurements was performed
using the ionophore nigericin (Sigma, St. Louis, MO) in buffers of known pH (26).
Doxorubicin Uptake and Distribution in Cells
Doxorubicin distribution in cells was evaluated by fluorescence microscopy. Cells
attached to a chambered cover glass were pretreated with pantoprazole (1mM) for 2 hours and
then incubated in media containing 2μg/ml doxorubicin for 1 hour. The drugs were washed out
and the fluorescence signal was recorded with excitation at 514nm and emission at 488nm using
a Zeiss Axiovert 200M fluorescence inverted microscope and the fluorescence signals were
captured with a Roper Scientific CoolSnap HQ CCD camera. To visualize endosomes, the cells
were exposed to the pH-sensitive endosomal dye, LysoSensor Yellow/Blue DND-160
(Molecular Probe, Inc. Eugene, OR) at a concentration of 5μM for 15 min. The fluorescent
signal was measured with excitation at 360nm and emission at 420nm.
To determine net uptake of doxorubicin in EMT-6, MCF-7 and A431 cell lines, 106 cells
were treated in vials with either saline or pantoprazole (1mM). After 2 hours, doxorubicin
(1.8µM) was added to each vial and incubated for 1 hour and then washed twice with PBS.
Mean doxorubicin fluorescence was measured using the Becton Dickinson FACScan (Franklin
Lakes, NJ), and Cell Quest software. The FL1 filter (530nm) was used to detect doxorubicin
fluorescence.
Doxorubicin Penetration in Multilayered Cell Cultures
Drug penetration was evaluated in an in vitro tumor-like environment using MCCs (27,
28). Approximately 2 x 105 cells were seeded on collagen-coated microporous Teflon
7
membranes (Millipore, Bedford, MA) and given 4-6 hours to attach. Experiments using MCCs
were carried out using EMT-6 and MCF-7 cells. A431-derived MCCs were not used because
cells did not grow well in these conditions. The membranes were immersed in α-MEM media in
a large vessel with continuous stirring and allowed to incubate at 37°C for approximately 6-8
days; this led to the formation of MCCs containing approximately 5 x 106 cells. The MCCs were
incubated in either media alone or media containing 1mM pantoprazole for 2 hours. The
penetration experiments were performed at 37°C in an atmosphere of 95% air and 5% CO2. In
order to evaluate doxorubicin penetration, solutions containing 10µmol/L radiolabeled
doxorubicin were prepared in 2 x α-MEM (without FBS) and mixed in a 1:1 ratio with 1% agar
solution; the agar is added to prevent convection. A volume of 0.5mL of this mixture was added
to one side of the MCC. The membranes were then floated in glass polyshell vials containing
18mL of α-MEM media. A cell-free membrane insert was included in all experiments as a
control. The drug, after penetrating through the MCC, appeared in the compartment below and
150µL samples were taken from this compartment over time and assessed by liquid scintillation
counting. 3H-sucrose was used as an internal standard at a concentration of 2µmol/L to ensure
minimal inter-experimental variations of the MCC thickness.
Fluorescent micrographs of MCCs were generated after exposure to doxorubicin, which
is fluorescent. After 1 hour of incubation, MCCs were removed and frozen, 10μm sections were
imaged using an Olympus Upright BX50 microscope with a 100W HBO mercury light source
equipped with 530 to 560 nm excitation and 573 to 647 nm emission filter sets. Images were
pseudo-colored using Image Pro Plus software.
8
Toxicity studies in mice
Mice were treated with either phosphate-buffered saline containing CaCl2 and MgCl2 alone,
doxorubicin alone (8mg/kg), or pantoprazole at each of the following doses: 100, 150, 200, 250,
300 mg/kg, alone and 2 hours prior to doxorubicin. The body weight of the mice was recorded
every other day.
Plasma concentrations of pantoprazole in mice
At various times after mice were treated with pantoprazole (200mg/kg), blood was
collected using cardiac puncture in heparin-coated tubes on ice, after sample collection the mice
were killed. Plasma was isolated and frozen by centrifugation. Pantoprazole concentration was
determined using high-performance liquid chromatography (HPLC) using a protocol similar to
that described by Peres et al. (29). Shimadzu SIL-20-AC auto-injector and Shimadzulc-20AD
pumps were used for HPLC-MS/MS analysis and Applied Biosystem MDS Sciex (Concord,
Ontario, Canada) API3200 tandem mass spectrometry equipped with a TurboIonSpray interface
was used subsequently. The data were processed using Analyst 1.4.2 software (Applied
Biosystem MDS Sciex).
Doxorubicin Distribution in Solid Tumors
Mice bearing EMT-6 or MCF-7 subcutaneous tumors in both flank regions were divided
randomly into groups of five and were treated when the mean tumor diameter was in the range of
8-12 mm. Animals were treated with phosphate-buffered saline containing CaCl2 and MgCl2,
doxorubicin alone, or pantoprazole prior to doxorubicin. Doxorubicin was given intravenously
at a dose of 25mg/kg to facilitate detection and quantification of drug auto-fluorescence.
Pantoprazole was administered i.p. two hours prior to doxorubicin treatment at a dose of 200
mg/kg. To detect hypoxia, EF5 was injected i.p approximately two hours prior to killing the
9
mice (0.2mL of a 10mM stock per mouse), and to detect functional blood vessels the perfusion
marker DiOC7 (1 mg/kg) was injected i.v.1 minute prior to killing the mice. Mice were killed 10
minutes after doxorubicin injection and the tumors were excised. Previous studies in our
laboratory have showed that doxorubicin is maximally distributed in solid tumors between 10
minutes and 3 hours after administration (6). Also, removing the tumor 10 minutes after
doxorubicin administration ensures an adequate concentration of pantoprazole in the plasma.
The tissues were embedded immediately in OCT compound, frozen in liquid nitrogen, and stored
at -70°C. Cryostat sections 10 μm thick were cut at 3 levels approximately 100 μm apart from
each tumor, mounted on glass slides.
Doxorubicin fluorescence was quantified with an Olympus Upright BX50 microscope
with a 100W HBO mercury light source equipped with 530-560 nm excitation/573-647 nm
emission filter sets. Tissue sections were imaged with a Photometrics CoolSNAP HQ2
(monochrome for fluorescence imaging) camera and tiled using a motorized stage so that the
distribution of doxorubicin was obtained for the entire tissue section. All images were captured
in 8-bit signal depth and subsequently pseudo-colored.
Tumor sections were first imaged for doxorubicin and the perfusion marker DiOC7 using
a TRITC and FITC filter set, respectively. Sections were then stained for blood vessels using
antibodies specific for the endothelial cell marker CD31 [rat anti-CD31 primary antibody
(1:100); BD Biosciences, and C73-conjugated goat anti-rat IgG secondary antibody (1:400)],
hypoxic regions were identified using a Cy5-conjugated mouse anti-EF5 antibody (1:50). Tumor
sections were imaged for CD31 using the Cy3 (530-560 nm excitation/573-647 nm emission)
filter set, and EF5 using the Cy5 far-red filter set. Image analysis was performed as previously
described in our laboratory (30), where doxorubicin 8-bit grayscale images were overlaid with
10
binary CD31 images. Non-functional vessels from the CD31 image were determined by
comparing the image with a binarized DiOC7 image and these vessels were removed. The
overlaid images were then run through a customized algorithm to generate drug intensity
distributions in relation to distance from the nearest blood vessel.
Tumor vasculature was quantified using Media Cybernetics Image Pro PLUS Software.
The total number of blood vessels was measured by setting a threshold for CD31 positive pixel
intensity and minimum blood vessel area (62 μm2) and counting the number of objects within
these settings. Objects above this threshold range but below the minimum area were removed as
artifacts (31). The tumor area was recorded and areas of necrosis and artifact were excluded.
The mean number of total blood vessels per tumor area was determined. The number of
functional blood vessels per tumor area was calculated in a similar manner using DiOC7 positive
pixels.
Growth delay studies
Two perpendicular diameters of tumors growing in the flanks of mice were measured
with a caliper and treatment began once tumors reached a diameter of 5-8 mm. Tumor volume
was estimated using the formula: 0.5(ab2), where a is the longest diameter, and b is the shortest
diameter.
To determine the effects of pantoprazole, mice bearing either MCF-7 or A431 tumors
were divided into four groups of 4-5 mice each and treated with either saline, doxorubicin alone
(8mg/kg i.v.), pantoprazole alone (200mg/kg i.p.) or pantoprazole 2 hours prior to doxorubicin
(200mg/kg + 8 mg/kg). For evaluation of growth delay following multiple doses of drugs, mice
were treated with either doxorubicin alone (6mg/kg i.v.), pantoprazole alone (200mg/kg i.p.) or
pantoprazole combined with doxorubicin (200mg/kg + 6 mg/kg), once a week for three weeks.
11
Every 2-3 days the tumor volume and body weight were measured. Measurements were taken
until tumors reached a maximum diameter of 1.2cm or began to ulcerate, when mice were killed
humanely. All mice were ear tagged and randomized to avoid bias with measurements. Growth
delay experiments were also done in EMT-6 tumors but due to the fast-growing nature of these
tumors, ulcerations and overgrown tumors prevented data from being collected beyond 8 days.
Statistical analysis
A one-way ANOVA, followed by a post hoc t-test were performed to determine
statistical differences between treatment groups. For drug uptake using flow cytometry and
quantification of functional tumor vasculature and hypoxia, t-tests were performed to determine
significant differences between treatment groups. P<0.05 was used to indicate statistical
significance.
Results
Proton pump inhibitors increase endosomal pH in tumor cells
Pantoprazole, lansoprazole or tamoxifen led to concentration-dependent increases in
endosomal pH in EMT-6 cells; increases in endosomal pH were observed with concentrations of
tamoxifen above 10μM, and with lansoprazole and pantoprazole at concentrations above 200μM
(Figure 1A). Similar effects were observed following exposure of MCF-7 cells to pantoprazole;
doxorubicin alone had a small effect to raise endosomal pH and enhanced the concentrationdependent effects of pantoprazole to increase endosomal pH (Figure. 1B).
Pantoprazole pretreatment influences doxorubicin uptake in tumor cells in culture
Photomicrographs of fluorescence in MCF-7 and EMT-6 (not shown) cells exposed to
doxorubicin alone show the drug to be present in the nucleus and in punctuate compartments
12
within the cytoplasm; and localized staining of doxorubicin with lysosensor gives evidence of
endosomal sequestration (Figure 2A). Treatment with pantoprazole reduces the amount of
doxorubicin fluorescence in the cytoplasm while it is retained within the nucleus (Figure 2A,
panel iii).
Doxorubicin uptake within cells pretreated with either saline or pantoprazole 1mM) was
measured using flow cytometry. In EMT-6 cells, doxorubicin fluorescence significantly
increased with pantoprazole pretreatment (p<0.05). In contrast, MCF-7 and A431 (not shown)
cells showed a decrease in doxorubicin fluorescence by 24% (p<0.05) and 36% (p<0.05),
respectively, with pantoprazole pretreatment (Figure 2B). When cells were treated with a lower
concentration of pantoprazole (100µM), similar changes in distribution were observed, but these
results were not significant (data not shown).
Doxorubicin penetration in multilayered cell cultures pretreated with pantoprazole
When MCCs were pretreated with 1mM pantoprazole there was a greater than 2-fold
increase in doxorubicin penetration for those grown from EMT-6 cells (p<0.05) (Figure 3A) and
~1.3-fold increase through those grown from MCF-7 cells (p<0.02) (Figure 3B). Internal
standards indicated by 3H-sucrose penetration were used to control for variations in thickness of
the cell cultures, and showed < 10% variation.
Photomicrographs of MCCs derived from MCF-7 cells indicate an increase in
doxorubicin fluorescence in cells more distal from the source of drug that are pretreated with
pantoprazole compared to control MCCs at 2 hours after the start of doxorubicin exposure
(Figure 3C,D). Similar changes in fluorescence were observed in MCCs derived from EMT-6
cells (photomicrographs not shown).
13
Plasma concentration of pantoprazole and toxicity in mice
After i.p. injections of 200mg/kg pantoprazole in mice, the peak plasma concentration
was ~300μM within the first hour after administration, and ~150 μM after 2 hours. After 5 hours
the concentration in the plasma decreased to less than 1% of the maximum concentration in the
blood and by 24 hours less than 0.01 μM of pantoprazole was detectable (Figure 4).
Toxicity studies in mice
Mice treated with saline alone showed a gradual increase in body weight over 20 days.
Mice treated with doxorubicin alone (8mg/kg iv) or pantoprazole alone (up to 300mg/kg)
showed minimal increase in body weight. Combined treatment led to no change in body weight
at pantoprazole doses of 100 or 150mg/kg, but at 200mg/kg mice showed a temporary decrease
in body weight (~15%) within the first 5-8 days after treatment followed by rapid recovery to
their original body weight. Mice treated with 250 or 300 mg/kg of pantoprazole and then
doxorubicin showed continual loss in body weight beyond 20 days (data not shown). The
maximum tolerated dose was determined to be 200mg/kg when combined with doxorubicin.
Effect of pantoprazole on distribution of doxorubicin in tumors
Photomicrographs taken from MCF-7 tumors show substantial increases in doxorubicin
fluorescence in tumors in mice pretreated with pantoprazole, compared to mice treated with
doxorubicin alone (Figure 5A, B).
Doxorubicin distribution was quantified in EMT-6 and MCF-7 tumors in mice at 10
minutes after injection. Doxorubicin fluorescence intensity was determined in relation to distance
to the nearest functional blood vessel in areas of interest with similar numbers of functional
vessels. In all tumors, there were steep gradients of decreasing doxorubicin fluorescence in
relation to distance from the nearest functional blood vessel in the section. In MCF-7 tumors,
14
there was a shallower gradient of decrease in doxorubicin distribution in the tumors pretreated
with pantoprazole compared to the tumors treated with doxorubicin alone (p<0.05) (Figure 5C).
However, in EMT-6 tumors there was no significant difference in drug distribution between
treatment groups (data not shown).
Pantoprazole pretreatment increases growth delay in MCF-7 tumors
Doxorubicin alone led to growth delay of MCF-7 tumors (p<0.05), and there was no
effect of pantoprazole alone (Figure 6A). Mice treated with pantoprazole prior to a single dose
of doxorubicin significantly increased tumor growth delay compared with the other treatment
groups (p<0.05), with minimal tumor growth out to 42 days; due to ulceration, mice were killed
thereafter. Studies were also carried out in a second human xenograft model that was derived
from the epidermoid carcinoma cell line A431: this tumor was resistant to doxorubicin but
pretreatment with pantoprazole led to a small increase in growth delay compared to groups
treated with the control (p=0.06) or doxorubicin alone (p=0.05) (Figure 6B). There was no
substantial loss in body weight after treatment (data not shown).
Mice treated with multiple doses of pantoprazole and doxorubicin (once a week for three
weeks), showed even greater growth delay of MCF-7 xenografts compared to the single dose
combination (Figure 6C). In A431 xenografts however, multiple doses of pantoprazole and
doxorubicin did not lead to significant growth delay (Figure 6D).
Discussion
The acidic microenvironment in solid tumors may cause resistance to some anticancer
drugs. The pH gradient between the cytoplasm and intracellular organelles may be modified in
cancer cells and there may be sequestration of basic drugs in acidic organelles (16). Proton
15
pump inhibitors increase pH in acidic vesicles such as endosomes (22), thereby inhibiting the
accumulation of basic cytotoxic agents in acidic organelles (32) and they may lead to apoptosis
of cancer cells (33).
In the present study, we investigated the effect of pantoprazole on endosomal pH of
cancer cells. We showed that pantoprazole increased endosomal pH at concentrations of 200μM
or higher, and the effect of pantoprazole to raise endosomal pH was enhanced in the presence of
(basic) doxorubicin. In addition to pantoprazole, lansoprazole, another type of PPI, and
tamoxifen also showed an increase in endosomal pH likely caused by blocking acidification in
these intracellular compartments.
We observed increased uptake of doxorubicin in EMT-6 cells with pantoprazole
pretreatment in contrast to decreased uptake with the pretreatment in MCF-7 and A431 cells. It
is likely that EMT-6 cells have slightly higher levels of P-glycoprotein (PgP) compared to MCF7 cells (34, 35). Similar patterns of doxorubicin uptake were seen in EMT-6 cells and PgP
overexpressing AR1 and NCI cells (Supplementary A). This would explain the low uptake in
EMT-6 cells treated with doxorubicin alone compared to that in MCF-7 and A431 cells. It has
been shown that PPIs, including pantoprazole, inhibit PgP (36), and this may contribute to the
increase in drug uptake after pantoprazole pretreatment of EMT6 cells. There is evidence that
pantoprazole may enhance uptake of PgP substrates across the blood-brain barrier that expresses
Pgp: for example there was increased penetration of imatinib in the brain with pantoprazole
pretreatment (37). We did not measure doxorubicin concentration in mouse brain with and
without co-administration of pantoprazole, but we did not observe neurotoxicity at the doses
used in our experiments.
16
Proton pump inhibitors have been shown to increase the sensitivity of resistant tumor
cells to the cytotoxic effects of several drugs, including cisplatin, 5-flurouracil and vinblastine,
and increase the cytotoxicity of these agents in cells (22-24). In the MCF-7 cells we observed
consistently decreased doxorubicin uptake with pantoprazole pretreatment. In these cells, the
nucleus may be saturated with drug and excess concentrations are likely stored in acidic vesicles,
while in PPI-treated cells this sequestration will be reduced leading to a decreased net uptake of
drug. With decreased uptake of drug, there is more drug available to distribute to cells distal
from blood vessels. Our in vitro data show an increase in doxorubicin penetration in
pantoprazole-treated MCCs.
If pantoprazole decreases net drug uptake in cells close to blood vessels, there is likely to
be increased doxorubicin distribution to more distant cells and the gradient of decreasing
doxorubicin intensity will be significantly shallower. In MCF-7 xenografts we observed a
significant increase in doxorubicin distribution in relation to the nearest blood vessel following
treatment with pantoprazole, but this effect was not observed in EMT-6 tumors. This may again
be attributed to slightly higher levels of PgP in EMT-6 cells. Previous studies in our laboratory
have shown that PgP inhibitors may decrease doxorubicin distribution in solid tumors derived
from PgP-expressing cells, most likely because they increase drug uptake in cells close to blood
vessels, such that there is less drug available to penetrate to distal cells (30, 38).
Causes of drug resistance in tumors are both multifactorial and interconnected such that
overcoming one mechanism may not improve drug resistance. An effective strategy is likely to
influence several processes that contribute to resistance to give an overall therapeutic benefit.
The effect of pantoprazole in MCF-7 cells and xenografts may improve therapeutic efficacy by
influencing the net effect of different mechanisms of resistance.
17
Our HPLC data shows that pantoprazole concentrations decrease exponentially after 2-3
hours. By administering pantoprazole 2 hours prior to doxorubicin, we hypothesized that
pantoprazole would be in sufficient concentration to increase pH within intracellular organelles
and inhibit doxorubicin sequestration into these organelles. However, our HPLC data indicate
maximum levels of pantoprazole in the plasma of mice that are lower than the concentration of
pantoprazole required to raise endosome pH in the in vitro experiments. High concentrations of
pantoprazole may account for increases in doxorubicin penetration in MCCs, but in drugsensitive xenografts, lower in vivo concentrations of pantoprazole in combination with
doxorubicin can still alter drug distribution. After 2 hours, plasma concentrations of
pantoprazole are around 200μM. In drug uptake experiments using flow cytometry, we observed
that pantoprazole alters doxorubicin uptake into cells at 1mM and at 100 μM, albeit to a lesser
extent. The finding that lower doses of pantoprazole are effective in vivo might be due to the
role of the microenvironment and its effects on ion gradients between intracellular compartments
and between intracellular and extracellular pH. The extracellular pH is known to be low in
tumor regions distant from functional blood vessels, an effect that will inhibit cellular uptake of
basic drugs (11); the pH gradient between the cytoplasm and the extracellular fluid may be
inhibited by proton pump inhibitors, leading to improved uptake of basic drugs such as
doxorubicin, although this effect is likely to have limited therapeutic benefit if the concentration
of doxorubicin in such regions is low.
In addition to contributing to drug sequestration, acidic endosomes have been shown to
be important in the process of autophagy (39). Autophagy may be a survival mechanism of
nutrient deprived cells (40, 41). Our laboratory has preliminary data to indicate that
pantoprazole is an inhibitor of autophagy because of its role in altering the pH of acidic
18
compartments; this may be an additional mechanism through which proton pump inhibitors may
effect therapeutic efficacy (42).
The strategies of altering drug sequestration to improve drug distribution in tumors, and
of inhibiting autophagy may overcome drug resistance. Our studies of tumor growth delay in
mice bearing MCF-7 xenografts indicate a therapeutic advantage of using pantoprazole in
combination with doxorubicin. Multiple-dose treatments showed even greater effects to delay
growth of MCF-7 tumors. These experiments were repeated in another xenograft model using
A431 tumors. A431 xenografts were resistant to doxorubicin and pantoprazole pretreatment had
only minimal effects to improve growth delay. Preliminary studies in our laboratory indicate
that docetaxel has much greater effects to delay growth of this tumor and using biomarkers to
demonstrate the distribution of (non-fluorescent) docetaxel (43), we have shown recently that
pantoprazole pretreatment enhances both drug distribution and antitumor effects considerably
(44). These studies indicate that pantoprazole in combination with some chemotherapeutic agents
may have important antitumor effects.
Conclusions
Our findings suggest that pantoprazole increases endosomal pH in tumor cells and may
increase nuclear uptake of doxorubicin within these cells. Pantoprazole increases tissue
penetration of doxorubicin in MCCs and improves doxorubicin distribution from blood vessels in
solid tumors. Pantoprazole given prior to doxorubicin increases growth delay when given as
single or multiple doses to mice bearing MCF7 xenografts.
Our data, and those of others (22-24), suggest that pretreatment with pantoprazole may be
an effective strategy to improve the therapeutic efficacy of chemotherapy in some solid tumors.
Increased drug sequestration and limited drug distribution may have important roles in
19
multifactorial drug resistance and must be considered when developing novel therapeutics
treatment strategies.
The preclinical results described in this report led to funding by the Komen Foundation of
a phase I trial of escalating doses of pantoprazole given prior to doxorubicin; this trial has shown
that high dose pantoprazole (240mg) can be given intravenously prior to standard dose
doxorubicin without apparent increase in toxicity, and that high serum levels of pantoprazole
(~100uM) can be achieved in patients. Although the main goal of a phase I trial is to evaluate
tolerance and toxicity in patients who have exhausted standard therapy, responses were seen in
that trial. A phase II trial using pantoprazole with chemotherapy has been initiated.
Funding
This work was supported by a grant from the Canadian Institute for Health Research.
Abbreviations
V-H+-ATPases, Vacuolar-H+-ATPases; PPI, Proton Pump Inhibitors; MCC, Multilayered Cell
Culture; PgP, P-glycoprotein; HPLC, High Performance Liquid Chromatography
Competing Interests
The authors have no competing interests.
Author Contributions
KJP carried out the drug uptake studies, the drug penetration and drug distribution studies, the
HPLC experiments and the growth delay studies as well as drafting the manuscript. CL
conducted the endosomal pH studies and helped in the drug uptake studies. QT participated in
the HPLC studies and the growth delay studies. IFT conceived of the study, obtained funding to
support it, participated in its design and coordination and helped to draft and edit the manuscript.
All authors read and approved the final manuscript.
20
Acknowledgements
We would like to acknowledge Dr. Eric Chen for providing support with the HPLC analysis.
21
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Figure Legends
Figure 1.
Endosomal pH measurements in cancer cells. A. EMT-6 cells exposed to
varying concentrations of lansoprazole (), pantoprazole (), and tamoxifen
(). B. MCF7 cells treated with varying doses of pantoprazole in the presence
() or absence () of doxorubicin. Cells were exposed to the pH sensitive dye
Lysosensor and fluorescence was measured using flow cytometry (data represents
mean ± SE; n=3).
Figure 2.
Doxorubicin uptake in tumor cells in culture. Fluorescent micrographs show
alterations in intracellular doxorubicin distribution in cancer cells in culture with
pantoprazole pretreatment. (A) MCF-7 cells were treated with (i) doxorubicin, (ii)
lysosensor or (iii) pantoprazole prior to doxorubicin. (B) Doxorubicin
fluorescence in EMT-6, MCF-7 and A431 cells pretreated with either saline or 1
mM pantoprazole was measured using flow cytometry (data represents mean ±
SEM; n=3).
Figure 3.
The penetration of doxorubicin in multilayered cell cultures (MCCs)
derived from MCF-7 cells as a function of time. MCCs were pretreated with
either saline or pantoprazole (1mM) followed by 14C-doxorubicin (data represent
mean ± SEM; n=4) (A). Photomicrographs of fluorescent doxorubicin in MCCs
derived from MCF-7 cells. MCCs were pretreated with either saline (B) or
pantoprazole prior to doxorubicin treatment (C) (scale bars represent 100µm).
25
Figure 4.
Plasma concentration of pantoprazole at various times after administration
of 200mg/kg i.p. to Balb/C nude mice. Plasma levels of pantoprazole were
determined by a validated high performance liquid chromatography method (data
represents mean ± SEM; n=4).
Figure 5.
The distribution of doxorubicin fluorescence intensity in relation to the
nearest functional blood vessel in MCF7 xenografts. Mice were treated with
either doxorubicin alone (10 minutes)(A) or pantoprazole (2 hours) and
doxorubicin (B) prior to tumor excision. Photomicrographs show blood vessels
(red), functional blood vessels (yellow), hypoxic regions (green) and doxorubicin
(blue) (scale bar represents 100μm). Tumors were imaged and quantified using a
customized algorithm to show a doxorubicin fluorescence intensity in relation to
distance from the nearest functional blood vessel (C) (data represent mean ±
SEM; n=6).
Figure 6.
Effect of pantoprazole and doxorubicin treatment on tumor growth in mice.
Mice bearing MCF-7 or A431 xenografts were treated once (A and B) or once a
week for three weeks (C and D) with saline, doxorubicin alone, pantoprazole
alone, or pantoprazole pretreatment prior to doxorubicin. Tumor volume in mice
was measured every 2-4 days (data represent mean ± SEM; n=8).
26
A
7.0
Endosom
mal pH
6.5
6.0
5.5
5.0
4.5
4.0
1
10
100
1000
10000
1000
10000
Concentration (μM)
B
7.0
Endosomal pH
H
6.5
6.0
5.5
50
5.0
4.5
1
10
100
Concentration ( μ M )
KJP - Figure 1
A
i
ii
B
iii
180
160
DOX
PTP + DOX
Mean DOX Fluorescence
140
120
100
80
60
40
20
0
EMT-6
KJP - Figure 2
MCF-7
Cell Line
A431
Relative Penetrattion of Doxorubicin
A
E
0.3
DOX
0.25
PTP + DOX
0.2
0.15
F
0.1
0.05
0
0
2
4
Time ((hours))
B
C
E
F
KJP - Figure 3
6
8
Pantopraazole Concentraation (µM)
1000
100
10
1
0.1
0.01
0.001
0
5
10
Time (hrs)
KJP - Figure 4
15
20
25
B
A
DO
OX Fluorescence Intensity (i.u
u.)
C
14
DOX
PTP + DOX
12
10
8
6
4
2
0
0
20
40
60
80
Distance (um)
KJP - Figure 5
100
120
140
Mean Tumor V
Volume (mm3)
A
Mean Tumor V
Volume (mm3)
A431
MCF-7
B
1000
1000
100
Control
DOX
PTP
DOX+PTP
10
0
10
20
30
40
100
Control
DOX
PTP
DOX + PTP
10
0
50
Days
10
20
D
1000
Mean Tu
umor Volume (m
mm3)
C
Mean TTumor Volume ((mm3)
30
Days
1000
100
Control
PTP (x3)
DOX (x3)
DOX + PTP (x3)
10
0
10
KJP – Figure 6
20
30
Days
40
50
100
Control
PTP (x3)
DOX (x3)
DOX + PTP (x3)
10
0
10
Days
20
30