Role of multidrug resistance associated protein 4 (MRP4 ABCC4) in the resistance and toxicity of oxazaphosphorines

ROLE OF MULTIDRUG RESISTANCE-ASSOCIATED
PROTEIN 4 (MRP4/ABCC4) IN THE IN VITRO ACTIVITY
OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE



ZHANG JING
(B.Med, School of Medicine, Zhejiang University, P.R. China)



A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PHARMACY
NATIONAL UNIVERSITY OF SINGAPORE
2008

- i -
 ACKNOWLEDGEMENTS
I like to thank my former supervisor, Dr. Shufeng Zhou and my supervisor
Prof Ho Chi Lui, Paul and co-supervisor Prof Ng Ka-Yun, Lawrence, for their
great support, guidance and encouragement, for their invaluable assistance in the
planning and conducting of the project, and for their advice when difficulties were
encountered.
I also like to thank Prof Tan May Chin, Theresa of the Department of
Biochemistry, National University of Singapore for providing the MRP4
transfected HepG2 cell line, which was the focus of this project.

I like to acknowledge the technical assistance given by all laboratory
officers and students in my department and acknowledge the scholarship from the
National University of Singapore and the generous support of the National
University of Singapore Academic Research Funds.
Finally, I want to make a special acknowledgement to my family for their
great moral support.
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 PUBLICATIONS ARISING FROM THIS THESIS
Referred Journal Papers
1. Zhang J, Ng LK, and Ho PC. Interaction of Oxazaphosphorine Anticancer
Agents with Multidrug Resistance Associated Protein 4. Biochemical
Pharmacology (under revision).
2. Tian Q, Zhang J,
Chan E, Dun W, and Zhou SF. Multidrug resistance proteins
(MRPs) and implication in drug development. Drug Development Research 2005;
64(1):1-18.
3. Tian Q, Zhang J
, Tan TM, Chan E, Duan W, Chan SY, Boelsterli UA, Ho PC,
Yang H, Bian JS, Huang M, Zhu YZ, Xiong WP, Li XT and Zhou SF. Human
Multidrug Resistance Associated Protein 4 Confers Resistance to Camptothecin
Analogs. Pharmaceutical Research 2005; 22(11): 1837-1853.
4. Zhang J, Tian Q, Chan SY and Duan W, and Zhou SF. Insights into
oxazaphosphorine resistance and possible approaches to its circumvention. Drug
Resistance Updates 2005; 8(5): 271-297.
5. Zhang J
, Tian Q, Chan SY, Duan W, Li SC, Zhu YZ, and Zhou SF.
Metabolism and Transport of Oxazaphosphorines and the Clinical Implications.
Drug Metabolism Reviews 2005; 37 (4):611-703.
6. Tian Q, Zhang J
, Chan SY, Tan TM, Duan W, Huang M, Zhu YZ, Chan E, Yu

Q, Nie YQ, Ho PC, Li Q, Ng LK, Yang HY, Hong W, Bian JS, and Zhou SF.
Topotecan is a Substrate for Multidrug Resistance Associated Protein 4. Current
Drug Metabolism 2006; 7(1): 105-118.
- iii -
7. Zhang J, Tian Q, and Zhou SF. Clinical pharmacology of cyclophosphamide
and ifosfamide. Current Drug Therapy 2006; 1(1):55-84.
8. Zhang J, Tian Q, and Zhou SF. Reversers for oxazaphosphorine resistance.
Current Cancer Drug Targets 2006; 6(5): 385-407.
Published Conference Abstracts
1. Zhang J, Tian Q, and Zhou SF. Resistance profiles of multidrug resistance
associated protein 4 to anticancer drugs. 17
th
Singapore Pharmacy Congress, 30
June-3 July 2005, Singapore.
2. Tian Q, Zhang J
, Tan MQ, Chan E, Chan SY, and Zhou SF. Resistance
profiles of camptothecins in HepG2 cells with overexpression of MRP4. 17
th

Singapore Pharmacy Congress, 30 June-3 July 2005, Singapore.
3. Suhaiemi TN, Zhang J, and Zhou SF. Multidrug resistance associated protein
4 (MRP4) confers resistance to cyclophosphamide. 17
th
Singapore Pharmacy
Congress, 30 June-3 July 2005, Singapore.
4. Zhang J
, Tian Q, and Zhou SF. Resistance profiles of multidrug resistance
associated protein 4 to anticancer drugs. Inaugural AAPS-NUS Student Chapter
Symposium, 16 September 2005, Singapore.
5. Tian Q, Zhang J

, and Zhou SF. Multidrug resistance associated protein 4
confers resistance to camptothecins. Inaugural AAPS-NUS Student Chapter
Symposium, 16 September 2005, Singapore.
6. Tian Q, Zhang J, Tan MQ, Chan E, Chan SY, Duan W, and Zhou SF. Human
multidrug resistance associated protein 4 confers resistance to camptothecins. 1
st

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Postgraduate Congress of Faculty of Science of NUS, 21-22 September 2005,
Singapore
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 TABLE OF CONTENTS
 ACKNOWLEDGEMENTS I
 PUBLICATIONS ARISING FROM THIS THESIS II
 TABLE OF CONTENTS V
 SUMMARY IX
 LIST OF TABLES XII
 LIST OF FIGURES XIII
 LIST OF ABBREVIATIONS XV
CHAPTER 1 INTRODUCTION 1
1.1 MULTIDRUG RESISTANT ASSOCIATED PROTEINS (MRPS) 2
1.1.1 An overview of MRPs 2
1.1.2 The specific role of MRP4/ABCC4 11
1.2 CYCLOPHOSPHAMIDE AND IFOSFAMIDE 17
1.2.1 Clinical activity and mechanism of action of cyclophosphamide and ifosfamide 17
1.2.2 Pharmacokinetics and pharmacodynamics of cyclophosphamide and ifosfamide 20
1.2.3 Drug resistance to cyclophosphamide and ifosfamide 29
1.3 OBJECTIVES 33
CHAPTER 2 CONFIRMATION OF THE EXPRESSION AND
FUNCTION OF MRP4/ABCC4 TRANSFECTED HEPG2 CELLS 35

2.1 INTRODUCTION 35
2.2 MATERIALS AND METHODS 36
2.2.1 Chemicals 36
2.2.2 Cell Culture 37
2.2.3 Cytotoxicity assay in V/HepG2 and MRP4/HepG2 cells 37
2.2.4 Western blot analysis 38
2.2.5 Quantitative analysis of MRP4/ABCC4 expression by immunostaining 39
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2.2.6 Quantitative Real-time Polymerase Chain Reaction (PCR) 39
2.2.7 Statistical analysis 40
2.3 RESULTS 40
2.3.1 Cytotoxicity assay 40
2.3.2 Western blot analysis 43
2.3.3 Quantitative analysis of MRP4/ABCC4 expression by immunostaining 44
2.3.4 Quantitative Real-time PCR 45
2.4 DISCUSSION 46
CHAPTER 3 THE ROLE OF MRP4/ABCC4 ON THE CYTOTOXICITY
OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE IN HEPG2 CELLS 48
3.1 INTRODUCTION 48
3.2 MATERIALS AND METHODS 49
3.2.1 Chemicals 49
3.2.2 Cell Culture 50
3.2.3 Cytotoxicity assay in V/HepG2 and MRP4/HepG2 cells 50
3.2.4 Cytotoxicities of cyclophosphamide and ifosfamide in V/HepG2 and MRP4/HepG2
cells with different MRP4/ABCC4 inhibitors or GSH synthesis inhibitor 50

3.2.5 Cytotoxicities of cyclophosphamide and ifosfamide in V/HepG2 and MRP4/HepG2
cells with MRP4/ABCC4 inducer 51

3.2.6 Western blot analysis 51

3.2.7 Quantitatove analysis of MRP4/ABCC4 expression by immunostaining 51
3.2.8 Statistical analysis 51
3.3 RESULTS 52
3.3.1 Cytotoxicity assay 52
3.4 DISCUSSION 72
CHAPTER 4 THE EFFECT OF MUTANT MRP4/ABCC4 ON THE
CYTOTOXICITY OF CYCLOPHOSPHAMIDE AND IFOSFAMIDE IN
HEPG2 CELLS 76
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4.1 INTRODUCTION 76
4.2 MATERIALS AND METHODS 78
4.2.1 Chemicals 78
4.2.2 Cell culture 78
4.2.3 Cytotoxicity assay in V/HepG2 and MRP4/HepG2 cells 79
4.2.4 Statistical analysis 79
4.3 RESULTS 79
4.3.1 Cytotoxicity assay 79
4.4 DISCUSSION 86
CHAPTER 5 INDUCING POTENCY OF CYCLOPHOSPHAMIDE AND
IFOSFAMIDE ON THE MRP4/ABCC4 EXPRESSION 88

5.1 INTRODUCTION 88
5.2 MATERIALS AND METHODS 89
5.2.1 Chemicals 89
5.2.2 Cell culture 89
5.2.3 Western blot analysis 89
5.2.4 Quantitatove analysis of MRP4/ABCC4 expression by immunostaining 90
5.2.5 Real-time PCR 90
5.2.6 Statistical analysis 90
5.3 RESULTS 90

5.3.1 Western blot analysis 90
5.3.2 Quantitative analysis of MRP4/ABCC4 expression by immunostaining 94
5.3.3 Real-time PCR 96
5.4 DISCUSSION 98
CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS 102
6.1 CONCLUSIONS 102
6.2 THESIS ACHIEVEMENTS 105
6.3 FUTURE DIRECTIONS 106
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 BIBLIOGRAPHY 108
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 SUMMARY
Multidrug resistance associated protein 4 (MRP4/ABCC4) is an organic
anion pump capable of transporting nucleoside, nucleotide analogs and cyclic
nucleotide. Increased expression of MRP4/ABCC4 in tumor cells is associated
with resistance to various chemotherapeutic agents such as methotrexate (MTX),
topotecan and others. MRP4/ABCC4 is identified as the contributor of multidrug
resistance (MDR). The oxazaphosphorines including cyclophosphamide (CP) and
ifosfamide (IF), represent an important group of therapeutic agents due to their
substantial antitumor and immuno-modulating activity. Resistance to
oxazaphosphorines is a major clinical problem often resulting in therapeutic
failure. Detailed investigations aimed at identification of resistant proteins and
circumventing approaches of intrinsic drug resistance are thus warranted.
The aim of this study was to investigate the resistance profiles of
MRP4/ABCC4 to oxazaphosphorines including CP and IF in the absence and
presence of various MRP4/ABCC4 inhibitors or MRP4/ABCC4 inducers by using
the MRP4/ABCC4 overexpressing HepG2 cells. Overexpression of
MRP4/ABCC4 conferred significant resistance to CP and IF in the 48-hr drug-
exposure assays. In MRP4/ABCC4 overexpressing HepG2 cells, the presence of
the MRP4/ABCC4 inhibitors including diclofenac, MK571, and celecoxib

decreased the cytotoxicity of CP and IF in 48-hr exposure assay. In addition, the
presence of DL-buthionine-(S,R)-sulphoximine (BSO), the glutathione (GSH)
synthesis inhibitor, partially reversed the resistance to CP and IF in
MRP4/ABCC4 overexpressing HepG2 cells. Furthermore, clofibrate (CFB),
which was reported to be a MRP4/ABCC4 inducer in mice, was found to enhance
MRP4/ABCC4-mediated resistance to CP and IF. Moreover, this resistance to CP
and IF was enhanced in mutant MRP4/ABCC4 (F324A or F324W)
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overexpressing HepG2 cells when compared with MRP4/ABCC4 overexpressing
HepG2 cells. This demonstrates that CP and IF are highly possible substrates of
MRP4/ABCC4 and GSH may play an important role in the resistance to CP and
IF mediated by MRP4/ABCC4.
Oxazaphosphorines may also have effects on the expression of
MRP4/ABCC4. This was investigated by detecting MRP4/ABCC4 expression in
HEK293 cells and HepG2 cells after these cells were incubated with media
containing different concentration of oxazaphosphorines including CP and IF. The
MRP4/ABCC4 inducer CFB was used as a positive control. The present study
showed that the positive control CFB can up-regulate MRP4/ABCC4 expression
at protein level in HEK293 cells. In addition, CP significantly increased the
MRP4/ABCC4 expression at both protein level and mRNA level in HEK293 cells
at higher concentration, while IF significantly decreased the MRP4/ABCC4
expression at mRNA level at lower concentration only. It indicates that CFB can
modulate MRP4/ABCC4 in vitro; whereas CP is a MRP4/ABCC4 inducer at
higher concentration. Furthermore, Overexpression of MRP4/ABCC4 conferred
significant resistance to CFB in the 48-hr drug-exposure assays. The
MRP4/ABCC4 inhibitors MK571 and celecoxib partially reversed the resistance
caused by CFB. It suggests that CFB is a potential substrate of MRP4/ABCC4.
In summary, the present study demonstrated that MRP4/ABCC4 plays an
important role in the in vitro activity of oxazaphosphorines. In addition, this study
suggests that CFB is a potential substrate of MRP4/ABCC4 and a modulator of

MRP4/ABCC4 in vitro. Further studies are needed to explore the effect of
MRP4/ABCC4 on the transport of oxazaphosphorines and CFB. It would also be
interesting to investigate the effect of the combination of oxazaphosphorines and
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MRP4/ABCC4 inhibitors on cancer treatment, and to investigate the effect of
MRP4/ABCC4 on the toxicity of CFB.

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 LIST OF TABLES
Table 1-1. Reported Substrates and Inhibitors for MRPs 9
Table 2-1. Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector
only to some MRP4/ABCC4 substrates. 41
Table 3-1. Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector
only to cyclophosphamide (CP) and ifosfamide (IF) with or without BSO and
some known MRP4/ABCC4 inhibitors. The cells were preincubated with BSO
(200 µM) for 24 h, and celecoxib (50 µM), diclofenac (200 µM), or MK571 (100
µM) for 2 h. 55
Table 3-2. Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector
only to cyclophosphamide (CP) and ifosfamide (IF) with the presence of
MRP4/ABCC4 inducer. 61

Table 3-3. Drug sensitivity of HepG2 cells expressing MRP4/ABCC4 or vector
only to clofibrate without or with the presence of some known MRP4/ABCC4
inhibitors 67

Table 4-1. Drug sensitivity of HepG2 cells expressing mutant MRP4/ABCC4,
MRP4/ABCC4 or vector only to MRP4/ABCC4 substrate (MTX) after drug
exposure time of 4 hr. 80
Table 4-2. Drug sensitivity of HepG2 cells expressing mutant MRP4/ABCC4,
MRP4/ABCC4 or vector only to cyclophosphamide (CP) and ifosfamide (IF) after

drug exposure time of 48 hr. 83
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 LIST OF FIGURES
Figure 1-1. Metabolism of Cyclophosphamide 22
Figure 1-2. Metabolism of Ifosfamide 24
Figure 2-1. Cytotoxicity of MTX and bis-POM-PMEA in V/HepG2 (□) and
MRP4/HepG2 (■) cells when the drugs were incubated for 4 hr (MTX) or 48 hr
(bis-POM-PMEA). 42
Figure 2-2. Western blot analysis of MRP4/ABCC4 expression in Hep G2 cells.
Lane 1 shows the V/HepG2 cells and lane 2 shows the MRP4/HepG2 cells. The
molecular mass (kDa) is indicated. 43
Figure 2-3. Quantitative analysis of MRP4/ABCC4 expression by
immunostaining in Hep G2 cells. Column 1 shows the V/HepG2 cells and column
2 shows the MRP4/HepG2 cells. 44

Figure 2-4. Quantitative real time PCR analysis of MRP4/ABCC4 mRNA in
V/HepG2 cells and MRP4/HepG2 cells. MRP4/ABCC4 mRNA was quantified
using real time PCR analysis (SYBR green) standardizing against the endogenous
control GAPDH. Data were normalized to controls and expressed as fold change
ratio to control levels. 45
Figure 3-1. Cytotoxicity of cyclophosphamide (CP) and ifosfamide (IF) in
V/HepG2 (□) and MRP4/HepG2 (■) cells when the drugs were incubated for 48
hr. 53
Figure 3-2. Cytotoxicity of cyclophosphamide (CP) incubated for 48 hr in
V/HepG2 (□) and MRP4/HepG2 (■) cells with the presence of BSO (200 µM),
diclofenac (200 µM), celecoxib (50 µM), and MK571 (100 µM). 57
Figure 3-3. Cytotoxicity of ifosfamide (IF) incubated for 48 hr in V/HepG2 (□)
and MRP4/HepG2 (■) cells with the presence of BSO (200 µM), diclofenac (200
µM), celecoxib (50 µM), and MK571 (100 µM). 59
Figure 3-4. Cytotoxicity of cyclophosphamide (CP) incubated for 48 hr in

V/HepG2 (□) and MRP4/HepG2 (■) cells with the presence of clofibrate (CFB)
(200 µM and 400 µM). 62

Figure 3-5. Cytotoxicity of ifosfamide (IF) incubated for 48 hr in V/HepG2 (□)
and MRP4/HepG2 (■) cells with the presence of clofibrate (CFB) (200 µM and
400 µM). 63

Figure 3-6. MRP4/ABCC4 protein expression by western blot in V/HepG2 and
MRP4/HepG2 cells exposed to different concentration of clofibrate (CFB) for 6
days. The bar graph shows the quantification of band intensity. *p<0.05, **p<0.01,
significantly different from the control group 65
Figure 3-7. Quantitative analysis of MRP4/ABCC4 protein expression by
immunostaining in V/HepG2 and MRP4/HepG2 cells exposed to different
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concentration of clofibrate (CFB) for 6 days. **p<0.01 significantly different from
the control group. 65
Figure 3-8. Cytotoxicity of clofibrate (CFB) incubated for 48 hr in V/HepG2 (□)
and MRP4/HepG2 (■) cells without or with the presence of celecoxib (50 µM)
and MK571 (100 µM). 68
Figure 3-9. Cytotoxicity of some alkylating agents including melphalan, busulfan,
nimustine hydrochloride and mechlorethamine hydrochloride incubated for 48 hr
in V/HepG2 (□) and MRP4/HepG2 (■) cells. 71

Figure 4-1. Cytotoxicity of methotrexate (MTX) incubated for 4 hr in V/HepG2
(□), MRP4/HepG2 (■), Mutant (F324A) (░) and Mutant (F324W) (▓) cells. 81
Figure 4-2. Cytotoxicity of cyclophosphamide (CP) and ifosfamide (IF) incubated
for 4 hr in V/HepG2 (□), MRP4/HepG2 (■), Mutant (F324A) (░) and Mutant
(F324W) (▓) cells. 85

Figure 5-1. MRP4/ABCC4 protein expression by western blot in HepG2 cells

exposed to different concentration of cyclophosphamide (CP), ifosfamide (IF) or
clofibrate (CFB) for 6 days. Contro 1 and control 2 are the controls for
cyclophosphamide, ifosfamide (or clofibrate) respectively. The bar graph shows
the quantification of band intensity. 92
Figure 5-2. MRP4/ABCC4 protein expression by western blot in HEK293 cells
exposed to different concentration of cyclophosphamide (CP), ifosfamide (IF) or
clofibrate (CFB) for 6 days. Contro 1 and control 2 are the controls for
cyclophosphamide, ifosfamide (or clofibrate) respectively. The bar graph shows
the quantification of band intensity. *p<0.05, **p<0.01, significantly different
from the control group. 93
Figure 5-3. Quantitative analysis of MRP4/ABCC4 protein expression by
immunostaining in HEK293 cells exposed to different concentration of
cyclophosphamide, ifosfamide or clofibrate for 6 days. *p<0.05, **p<0.01
significantly different from the control group. 95
Figure 5-4. Quantitative real-time PCR analysis of MRP4/ABCC4 mRNA in
HEK293 cells exposed to different concentration of cyclophosphamide,
ifosfamide or clofibrate for 6 days. MRP4/ABCC4 mRNA was quantified using
quantitative real-time PCR (SYBR green method) standardizing against the
endogeneous control GAPDH. Data were normalized to GAPDH and expressed as
ratio to GAPDH levels (n=3). *p<0.05, **p<0.01 significantly different from the
control group. 97


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 LIST OF ABBREVIATIONS
4-OH-CP 4-hydroxy-CP
4-OH-IF 4-hydroxy-IF
6-MP 6-mercaptopurine
6-TG 6-thioguanine
ABC ATP-binding cassette

ADH Alcohol dehydrogenase
ALDH Aldehyde dehydrogenase
ANOVA One-way analysis of variance
AUC Area under the plasma concentration-time curve
AZT Azidothymidine
BAR Bile acid receptor
BCRP Breast cancer resistance protein
Bis-POM-PMEA Bis(pivaloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine
BSA Alubumin from bovine serum
BSEP Bile salt export pump
BSO DL-buthionine-(S,R)-sulphoximine
cAMP Cyclic adenosine monophosphate
cGMP Cyclic guanosine monophosphate
cMOAT Canalicular multispecific organic anion transporter
cPr-PMEDAP Cyclopropyl-PMEDAP
CAR Constitutive androstane receptor
CFB Clofibrate
CP Cyclophosphamide
CPT-11 Irinotecan
- xvi -
CYP Cytochrome P-450
DHEAS Dehydroepiandrosterone-3-sulfate
DMEM Dulbecco’s modified Eagle’s medium
DMSO Dimethyl sulphoxide
DNP-SG 2,4-dinitrophenyl S-glucuronide
E
2
17βG Estradiol 17-beta-D-glucuronide
FXR Farnesol X-activated receptor
GSH Glutathione

GST Glutathione S-transferase
HIV Human immunodeficiency virus
IF Ifosfamide
IL-6 Interleukin-6
i.v. Intravenous
LTC4 Leukotriene C4
MDR Multidrug resistance
MetIMP 6-methyl-tIMP
MK571 3-([(3-(2-[7-chloro-2-quinolinyl] ethenyl)phenyl)-((3-
dimethylamino-3-oxopropyl)-thio)methyl]thio) propanoic acid
MRP Multidrug resistance associated protein
MRP4/HepG2 HepG2 cells with stably transfected MRP4
MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazonium bromide
MTX Methotrexate
NBD Nucleotide binding domain
NBMPR Nitrobenzyl mercaptopurine riboside
NEM-GS N-ethylmaleimide S-glutathione
- xvii
-
Nrf2 Nuclear factor-E2-related factor 2
NSAIDs Nonsteroidal antiinflammatory drugs
OAT Organic anion transporter
PAH p-Aminohippurate
PBS Phosphate buffered saline
PCR Polymerase Chain Reaction
PgP P-glycoprotein
PG Prostaglandin
PMEA 9-(2-phosphonylmethoxyethyl)adenine
PMEDAP 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine
PMEG 9-(2-phosphonomethoxyethyl)guanine

PPARα Peroxisome proliferator-activated receptor α
SD Standard deviation
S.E.M Standard error of the mean
TCPOBOP 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene
tIMP Thioinositol monophosphate
TEMED N,N,N′,N′-Tetramethylethylenediamine
TNF-α Tumor necrosis factor alpha
TXB2 Thromboxane B2
tXMP Thioxanthosine monophosphate
V/HepG2 HepG2 cells with insertion of vector only

- 1 -
CHAPTER 1 INTRODUCTION
Conventional chemotherapy aims to kill or disable tumor cells by direct or
indirect mechanisms, while preserving the normal cells [1]. Many chemotherapeutic
agents (e.g. Vinca alkaloids, oxazaphosphorines, camptothecins, etc) are developed to
treat many different malignancies. However, conventional chemotherapy is only
successful when the cancer is detected at its early stage, or is limited to certain types
of cancer (e.g. leukemia). Most chemotherapeutic agents can cause dose-limiting
toxicities which may be transient toxicity, or have long-term effects on the lungs,
heart and reproductive organs causing permanent organ damage, secondary tumor (e.g.
leukemia and brain tumor), or death. Increasing the selectivity of chemotherapeutic
agents may reduce dose-limiting toxicities, and coadministration of multiple
chemotherapeutic agents has become standard regimen for the treatment of nearly all
carcinomas and haematological malignancies. In addition, the major cause of failure
of antitumor chemotherapy is the development of multidrug resistance (MDR). Tumor
cells may have intrinsic resistance to current chemotherapeutic agents or develop
resistance after exposure. Cellular mechanisms of MDR include defective drug
transport (reduced drug transport or increased drug efflux), altered drug activation or
inactivation, and/or enhanced repair or tolerance to DNA damage.

Multidrug resistance associated proteins (MRPs) are believed to contribute to
the development of MDR. Therefore, knowledge on structures and functions of MRPs
is important to develop successful approaches on reversion of MDR. The rest of this
chapter will present an overview on structures and functions of MRPs, the specific
role of MRP4/ABCC4 in chemotherapy, mechanism of action of oxazaphosphorines,
pharmacokinetics and pharmacodynamics of oxazaphosphorines, drug resistance to
oxazaphosphorines and concludes with objectives of this research.
- 2 -
1.1 MULTIDRUG RESISTANT ASSOCIATED PROTEINS (MRPs)
MDR is the phenomenon in which cells show simultaneous resistance to
several different structurally and functionally unrelated drugs that do not have the
same mechanism of actions. P-glycoprotein (PgP/ABCB1) is the first drug efflux
pump identified as the contributor of MDR [2]. It is a 170 kDa plasma glycoprotein
encoded by

human MDR1 gene, which belongs to the ATP-binding cassette (ABC)
family of transporters. Such protein is expressed constitutively in a number of normal
tissues with high levels on the apical surfaces of epithelial cells in the liver (bile
canaliculi), kidney (proximal tubule), and small and large intestine (columnar mucosal
cell) [3]. Furthermore, PgP/ABCB1 can cause considerable resistance to a number of
chemotherapeutic agents, including anthracyclines (e.g. daunorubicin and doxorubicin)
[4], Vinca alkaloids (vincristine and vinblastine) [5], paclitaxel [6], and camptothecins
[7]. In addition to PgP/ABCB1, the mitoxantrone resistance gene, MXR, also known
as the breast cancer resistance protein (BCRP/ABCG2) is a potent contribtor of
MDR[8]. BCRP/ABCG2 has the ability to confer high levels of resistance to
anthracyclines (e.g. daunorubicin and doxorubicin) [9], mitoxantrone [9], bisantrene
[8], and the camptothecins (e.g. topotecan and SN-38) [10, 11]. Other than
PgP/ABCB1 and BCRP/ABCG2, many different transporters can confer the
resistance to clinically important anticancer agents. Almost all transporters belong to
MRP family, which is a subfamily of ABC transporters.

1.1.1 An overview of MRPs
All MRP members have hydrophobic transmembrane domains and
cytoplasmic nucleotide binding domains (NBDs) [12]. The NBDs are responsible for
the ATP binding/hydrolysis that drives drug transport [13]. The transmembrane
- 3 -
domains contain the drug-binding sites, which are likely to be located in a flexible
internal chamber that is sufficiently large to accommodate different drugs. MRPs can
be categorized according to the presence or absence of a third (NH
2
-terminal)
membrane-spanning domain in their structure. This topological feature can be found
in MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6 and
MRP7/ABCC10, while it is not found in MRP4/ABCC4, MRP5/ABCC5,
MRP8/ABCC11 and MRP9/ABCC12. MRPs with this structural feature have the
ability to transport conjugates, while MRPs without it are able to transport cyclic
nucleotides. Various MRPs show considerable differences

in their tissue distribution,
substrate specificities, and proposed

physiological and pharmacological functions.
MRP1/ABCC1 was found in 1992 in lung cancer H69AR cell line conferring
resistance to many chemotherapeutic agents, which had been demonstrated not to
overexpress PgP/ABCB1 previously [14, 15]. MRP1/ABCC1 is nearly present in all
major tissues and in all peripheral blood cell types. MRP1/ABCC1 is capable of
conferring resistance to several families of natural product drugs like PgP/ABCB1,
including Vinca alkaloids (vincristine and vinblastine), anthracyclines (e.g.
daunorubicin and doxorubicin), epipodophyllotoxins (etoposide) [16], and
camptothecins (irinotecan (CPT-11) and SN-38) [17]. In addition, the fluorescent
probe calcein [18, 19], the human immunodeficiency virus (HIV) protease inhibitors

(saquinavir and ritonavir) [20], the antiandrogen flutamide and its active metabolite
hydroxyflutamide [21], and the organic arsenic as a triglutathione conjugate [22] are
transported by MRP1/ABCC1. Furthermore, MRP1/ABCC1 is able to transport
different structural glutathione (GSH), glucuronate and sulfate conjugates, including
estradiol 17-β-D-glucuronide (E
2
17βG), the cysteinyl leukotriene C
4
(LTC
4
), and
sulfated bile acids [23, 24]. Substrates of MRP1/ABCC1 also include neutral and
- 4 -
basic cytotoxic compounds without conjugation with GSH or other anionic drugs [25,
26]. However, intracellular GSH is needed when MRP1/ABCC1 transports these
chemicals [27, 28]. Moreover, various inhibitors that inhibit MRP1/ABCC1 transport
activity have been described. Some inhibitors such as probenecid, sulfinpyrazone,
indomethacin and benzbromarone can modulate the activity of many different
transporters including organic anion transporters(OATs) that do not belong to the
ABC family [29]. Some inhibitors that are relatively specific to the MRP-related
transporters are sulphonylurea, glibenclamide [30], ONO-1078, and MK571 [31].
Many inhibitors are dual inhibitors of MRP1/ABCC1 and PgP/ABCB1. These include
PAK-104P [32], verapamil and cyclosporin A [25], and several bioflavonoids
(genistein and quercetin) [19]. In addition, LY475776 is a highly specific and potent
MRP1/ABCC1 inhibitor in a GSH-dependent manner [33]. Recently, the flavonoid
myricetin showed the ability to modulate MRP1/ABCC1 mediated resistance to the
anticancer drug vincristine in MRP1/ABCC1 transfected MDCKII cells [34].
MRP2/ABCC2 is also known as the canalicular multispecific organic anion
transporter (cMOAT). Mutations of the MRP2/ABCC2 gene result in the Dubin-
Johnson syndrome which is characterized by conjugated hyperbilirubinemia and

impaired hepatobiliary secretion of a wide range of amphipathic compounds [35].
MRP2/ABCC2 has shown resistance to a broad range of natural product drugs,
including etoposide, Vinca alkaloids (vincristine and vinblastine), anthracyclines (e.g.
daunorubicin and doxorubicin) [36], and camptothecins [7]. It seems that
MRP1/ABCC1 and MRP2/ABCC2 have some substrates in common. However, there
are some important differences. With respect to anticancer agents, MRP2/ABCC2
functions as a cellular cisplatin [37] and taxanes (paclitaxel and docetaxel) [38]
transporter. In addition, MRP2/ABCC2 efficiently transports the HIV protease
- 5 -
inhibitors including saquinavir, indinavir and ritonavir [39]. Both MRP1/ABCC1 and
MRP2/ABCC2 transport many of the glutathione, glucuronate, and sulfate conjugates,
but the affinities of the two transporters can differ significantly. For example,
bilirubin mono- and bis-glucuronides are better substrates for MRP2/ABCC2,
whereas MRP1/ABCC1 has higher affinity for LTC
4
[40].

Inhibitors of
MRP2/ABCC2 have been described. Recently, in vitro studies showed that the
flavonoids including robinetin and myricetin appeared to be good inhibitors for
MRP2/ABCC2 [41]. In addition, the non-nucleoside reverse transcriptase inhibitors
including delavirdine and efavirenz, and the nucleoside reverse transcriptase inhibitor
emtricitabine showed a significant and concentration-dependent inhibition of
MRP1/ABCC1, MRP2/ABCC2 and MRP3/ABCC3 [42].
Among the MRP family, MRP3/ABCC3 has 58% and 48% amino acid
sequence resemblance with MRP1/ABCC1 and MRP2/ABCC2 [43]. MRP3/ABCC3
is mainly detected in the liver, colon, intestine, and prostate at the mRNA level and in
adrenal gland, kidney, colon, pancreas, gallbladder, and liver at the protein level [44,
45]. As an OAT like MRP1/ABCC1 and MRP2/ABCC2, MRP3/ABCC3 confers
resistance to some anticancer drugs. However, the substrate profile of MRP3/ABCC3

is narrow and limited to vincristine, methotrexate (MTX), and epipodophyllotoxins
(teniposide and etoposide) [46, 47]. Overexpression of MRP3/ABCC3 is specifically
associated with increased ATP-dependent transport of E
2
17βG, 2,4-dinitrophenyl S-
glucuronide (DNP-SG), and LTC
4.
The transport of E
2
17βG by MRP3/ABCC3 was
inhibited by both etoposide and methotrexate in a concentration-dependent manner
[48]. In addition, MRP3/ABCC3 may function as a backup detoxification system for
liver cells if normal canalicular route is damaged by cholestatic diseases.
MRP3/ABCC3 can recognize the monoanionic bile acids such as glycocholate and
- 6 -
taurocholate, which is distinguished from other MRP family members [49, 50].
Recently, Zamek-Gliszczynski MJ et al. reported that MRP3/ABCC3 mediates the
hepatic basolateral excretion of diverse glucuronide conjugates in MRP3/ABCC3
knock out mice [51].
MRP5/ABCC5 is able to transport cyclic adenosine monophosphate (cAMP)
and cyclic guanosine monophosphate (cGMP), but with differential affinity [52].
MRP4/ABCC4 has a higher affinity for cAMP than that of MRP5/ABCC5, whereas
MRP5/ABCC5 has a higher affinity for cGMP than that of MRP4/ABCC4 [53]. The
transport of cyclic nucleotides by MRP4/ABCC4 and MRP5/ABCC5 has led to the
speculation that they might be involved in the regulation of the intracellular
concentration of these important second messengers. MRP5/ABCC5 has also shown
resistance to 9-(2-phosphonylmethoxyethyl)adenine (PMEA), 6-mercaptopurine (6-
MP) and 6-thioguanine (6-TG) [54]. In addition, MRP5/ABCC5 confers resistance to
5-fluorouracil and transports its monophosphorylated metabolites in vitro. This
resistance can be reversed by several inhibitors including probenecid, MK571,

sildenafil and trequinsin [55]. However, MRP5/ABCC5 shows no resistance to natural
anticancer compounds or MTX.
MRP6/ABCC6 is an amphipathic anion transporter able to transport
glutathione conjugates, such as LTC
4
and N-ethylmaleimide S-glutathione (NEM-GS),
and the cyclopentapeptide BQ123 which has a low affinity to E
2
17βG. Effective
inhibitors of MRP1/ABCC1 and MRP2/ABCC2, including indomethacin, probenecid,
and benzbromarone, can inhibit the MRP6/ABCC6-dependent NEM-GS transport
[56]. Additionally, MRP6/ABCC6 shows low level of resistance to several natural
product agents, including etoposide, teniposide, doxorubicin, and daunorubicin [57].
MRP6/ABCC6 is mainly present in the liver and kidney, besides in several other
- 7 -
organs with a low expression level [58]. MRP7/ABCC10 has the lowest amino acid
sequence identity with other MRP family members [59]. It is able to mediate the
transport of amphipathic anions, such as E
2
17βG [60]. In addition, MRP7/ABCC10 is
a resistance factor to several natural product anticancer agents including vincristine,
vinblastine, and taxanes, especially docetaxel [61]. However, MRP7/ABCC10 mRNA
is present in normal organs at very low levels.
MRP8/ABCC11 and MRP/ABCC12 have high amino acid sequence
resemblance with MRP4/ABCC4 and MRP5/ABCC5. MRP8/ABCC11 is mainly
present in normal breast and testis, while little is in the liver, brain, and placenta.
MRP8/ABCC11 has been characterized as an amphipathic anion transporter. It
participates in physiological processes involving the monoanionic bile acids
glycocholate and taurocholate, conjugated steroids such as LTC
4,

DNP-SG, E
2
17βG,
and dehydroepiandrosterone-3-sulfate (DHEAS), and cyclic nucleotides [62]. With
the ability to efflux cAMP and cGMP, MRP8/ABCC11 confers resistance to purine
and pyrimidine nucleotide derivatives [63]. MRP9/ABCC12 is a specific member of
MRP family, with two major transcripts of 4.5 and 1.3 kb. Because the larger 4.5-kb
is mainly expressed in breast tumor and seldom in normal breast, MRP9/ABCC12
may represent a novel target in the treatment of breast cancer [64]. However, the
functional characteristics of MRP9/ABCC12 are still unclear.
MRPs are capable of transporting a structurally diverse array of endo- and
xenobiotics and their metabolites across cell membranes. They play an important role
in the absorption, disposition and elimination of many therapeutic agents in the body.
In particular, increased expression of these drug transporters in tumor cells is
associated with resistance to a number of important chemotherapeutic agents. With
the accumulation of information on drug resistance profile and physiological function