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Studies on the mechanism and regulation of the transport of 5-fluorouracil (5FU) into tumor cells

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Studies on the mechanism and regulation of the transport of 5-fluorouracil (5FU) into tumor cells

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Content STUDIES ON THE MECHANISM AND REGULATION OF THE
TRANSPORT OF 5-FLUOROURACIL (5FU) INTO TUMOR CELLS
by
Chioma Jane Ikonte
A Dissertation Presented to the
FACULTY OF THE GRADUTE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
May 2001
Copyright 2001 Chioma Jane Ikonte
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UM I Number: 3027729
Copyright 2001 by
Ikonte, Chioma Jane
All rights reserved.
___ ®
UMI
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This dissertation, written by
under the direction of h.ltC. Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School, in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
Deart of Graduate Studies
Date
D ISSPPT A T T nM TTHUTUfTTTCT:
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Chioma Jane Ikonte Walter Wolf, Ph.D.
ABSTRACT
STUDIES ON THE MECHANISM AND REGULATION OF THE TRANSPORT
OF 5-FLUOROURACIL (5-FU) INTO TUMOR CELLS
Studies were performed to elucidate the mechanism and the regulation of the
transport of 5-Fluorouracil (5FU) into tumor cells using the Walker 256 adenocarcinoma
cells as the tumor model and 1 9 F NMR spectroscopy as the tool of analysis. The degree of
effectiveness of 5FU (an antineoplastic agent) has been correlated with the extent of its
uptake, retention and intracellular activation in tumors. A better understanding of the
mechanism and the factors that regulate its transport into tumor cells could allow the
identification of appropriate modulators that would selectively enhance uptake into tumor
cells.
Studies in rats bearing the Walker 256 tumors suggest a non-linear dose
dependence and a saturable process for the 5FU transport into tumors. A five-fold
increase in the administered dose of 5FU resulted in only a 50% increase in the amount of
free 5FU measured intracellularly. Studies on Walker 256 cell cultures suggest the
presence two transport mechanisms: a saturable carrier-mediated active transport process
and an equilibrative transport system for 5FU into tumor cells mediated through the
nitrobenzylthioinosine (NBTI) equilibrative-insensitive (ei) nucleoside transporter. 5FU
accumulated intracellularly against its concentration gradient in the presence and absence
1
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of extracellular Na+, was inhibited in the absence of extracellular glucose and in the
presence o f a glycolytic inhibitor. Structurally related nucleobases: uracil, thymine, and
nucleosides: uridine and cytidine competitively inhibited 5FU transport, as well as
dypridamole, a nucleoside transport inhibitor.
These studies also show evidence that the pH gradient imposed across the tumor
cell membrane affects the degree of transport of 5FU into the tumor cells. Methotrexate
(MTX) was identified as an effective modulator of both the anabolism and intracellular
transport of 5FU, with a 33% increase in the initial transport of 5FU after a 60-min pre-
incubation of cells with MTX.
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DEDICATION
FOR MY FAMILY AND WELL-WISHERS
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ACKNOWLEDGEMENTS
I would like to express my deepest appreciation and profound gratitude to my
advisor, Prof. Walter Wolf for giving me the opportunity to purse my studies to the
Ph.D. level, especially for the chance to carry out research aimed at making cancer a
disease of the past. His directions, challenges, support and personal enthusiasm made
this project a very interesting and rewarding experience for me. He provided me with
a solid foundation upon which to build my scientific research career.
I would also like to extend my sincere gratitude to Prof. Wei-Chang Shen for his
valuable support, guidance and insights that made this project both challenging and
interesting. I would also like to thank him for a space in his laboratory in which most
of the cell culture studies were carried out. I would also like to thank Prof. Austin
Mircheff, Dr. Ian Haworth and Dr. Sarah Ham-Alvarez for their excellent advice,
suggestions and instructions throughout the various stages of this work.
My sincere thanks to all members of my family who supported me throughout this
research endeavor, especially my husband, Dr. Chijioke Ikonte who was always
there for me throughout these studies. Thanks also to my mother, Mrs. Charity
Okpara for assisting me with the care of my children while I pursed my studies.
Finally, I would like to express my deepest gratitude to the National Cancer Institute
(NCI) of the National Institute of Health for the 3 year fellowship given to me that
largely supported me through my graduate studies. Likewise my sincere gratitude
goes to the American Association of Cancer Research (AACR) for the Minority
Scholar awards granted to for 3 years which enabled me to attend scientific meetings
that proved to be very valuable in my research work.
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CHAPTER PAGE
DEDICATION....................................................................................... ii
ACKNOWLEDGEMENT................................................ iii
TABLE OF CONTENTS.............................................. iv
LIST OF TABLES........................................................................... viii
LIST OF FIGURES.......................................................... xi
ACRONYMS............................................................................................ xv
1.0 INTRODUCTION.................................................................................... 1
1.1 5-FLUOROURACIL................. 4
1.1.1 What is 5FU?................................................................................ 4
1.1.2 CLINICAL USE OF 5FU............................................................ 5
1.1.3 METABOLISM OF 5FU............................................................. 6
1.1.3.1 THE ANABOLIC PATHWAY...................................... 6
1.1.3.2 THE CATABOLIC PATHWAY.................................... 7
1.1.4 MECHANISMS OF ACTION OF 5FU..................................... 9
1.1.5 MODULATION OF THE ACTION OF 5FU.......................... 10
1.2 TRANSPORT PROCESSES.................................................................. 12
1.2.1 TRANSPORT OF 5FU.................................................................. 12
1.2.2 NUCLEOBASE TRANSPORT SYSTEM...................................... 16
1.2.3 NUCLEODSIDE TRANSPORT SYSTEM................................ 19
1.2.4 MODULATION OF 5FU TRANSPORT BY METHOTREXATE
(MTX)................................................. 22
1.2.5 KINETICS OF SATURABLE TRANSPORT.............................. 23
1.2.5.1 MICHAELIS-MENTEN EQUATION........................... 23
1.3 ASSAY METHODS FOR STUDYING THE TRANSPORT OF 5FU
1.3.1 ANIMAL STUDIES................ 24
1.3.2 CELL CULTURE STUDIES... ..................................................... 25
1.3.3 NMR SPECTROSCOPY METHODS............................................. 27
1.3.3.1 1 9 F NMR SPECTROSCOPY ............ 29
1.3.3.2 EXPERIMENTAL PROCEDURE AND PROBLEMS ....... 29
1.3.3.3 EXPERIMENTAL MEASUREMENTS .............. 32
iv
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1.3.3.3.1 CHEMICAL SHIFT MEASUREMENT......................... 32
1.3.3.3.2 SENSITIVITY....................................... 32
1.3.3.4 QUANTITATION OF AN NMR SPECTRUM....................... 34
2.0 STATEMENT OF OBJECTIVE .................... 36
3.0 MATERIALS AND METHODS......................... 39
3.1 DRUGS AND CHEMICALS.................................................................... 39
3.2 WALKER 256 SOLID CARCINOMA FOR ANIMAL STUDIES 40
3.2.1 TUMOR TRANSPLANTATION. ........................................... 40
3.2.2. MAINTENANCE OF THE CELL LINE...................................... 41
3.3 WALKER 256 CARCINOMA CELLS FOR CELL CULTURE 41
3.3.1 SUB-CULTURE OF CELLS ................................................... 42
3.3.2 MAINTENANCE OF THE CELL LINE ........................... 43
3.4 EX-VIVO AND IN-VITRO STUDIES ............................ 44
3.4.1 TRANSPORT STUDIES USING ANIMAL MODELS 44
3.4.1.1 PERCHLORIC ACID EXTRACTION OF ANIMAL
TISSUES ........................................................................... 44
3.4.1.2 ANALYSIS OF THE ACID INSOLUBLE FRACTION.. 45
3.4.2 TRANSPORT STUDIES USING CELL CULTURES................. 45
3.4.2.1 A TYPICAL TRANSPORT STUDY USING CELL
CULTURES........................................................... 46
3.4.2.2 PERCHLORIC ACID EXTRACTION OF CULTURED
CELLS.................................................................... 47
3.4.2.3 ANALYSIS OF THE ACID INSOLUBLE FRACTION.. 47
3.4.2.4 ESTIMATION OF THE CELL PROTEIN CONTENT.... 48
3.5 ANALYTICAL METHOD: IN-VITRO 1 9 F NMR
SPECTROSCOPY. ........ 49
3.5.1 QUANTITATION FOR IN VITRO 1 9 F NMRS............................ 51
3.5.1.1 SIGNAL SATURATION FACTOR.............................. 51
3.5.1.2 PULSE WIDTH CALIBRATION................................. 51
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3.5.2 QUANTITATIVE CALIBRATION OF TISSUE EXTRACTS.. 52
3.5.3 QUANTITATIVE CALIBRATION OF CELL EXTRACTS .... 53
4.0 RESULTS
STUDIES ON THE MECHANISM OF TRANSPORT OF 5FU INTO
WALKER 256 CELLS ................. .......... 56
4.1 ANIMAL STUDIES
4.1.1 ACCUMULATION OF FREE 5FU IN THE TUMOR ASF............. 56
4.1.2 ACCUMULATION OF FREE 5FU IN THE LIVER A SF 60
4.2 CELL CULTURE STUDIES
4.2.1 ACCUMULATION OF FREE 5FU IN THE WALKER 256
TUMOR CELLS...................................................................... 64
4.3 RATIO OF INTRACELLULAR/EXTRACELLULAR 5FU 70
4.4 EFFECT OF EXTRACELLULAR Na+ ON 5FU TRANSPORT.... 72
4.4.1 5FU TRANSPORT IN THE ABSENCE OF
EXTRACELLULAR Na+ ............................................................... 72
4.4.2 EFFECT OF OUABAIN ON THE TRANSPORT OF 5FU 78
4.5 EFFECT OF GLUCOSE ON 5FU TRANSPORT............................ 80
4.5.1 5FU TRANSPORT IN THE ABSENCE OF
EXTRACELLULAR GLUCOSE .......................................... 80
4.5.2 EFFECT OF A GLYCOLYTIC INHIBITOR ON THE
TRANSPORT OF 5FU................... 84
4.6 EFFECT OF EXTRACELLULAR pH ON 5FU TRANSPORT 85
4.7 EFFECT OF METABOLIC INHIBITORS ON 5FU TRANSPORT................. 88
4.7.1 EFFECT OF 2,4-DINITROPHENOL ON 5FU TRANSPORT ...88
v i
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4.8 KINETICS OF 5FU UPTAKE 89
4.9 EFFECT OF STRUCTURAL ANALOGS 5FU TRANSPORT 90
STUDIES ON THE NATURE AND SPECIFICITY OF THE 5FU
TRANSPORTER (S)
4.10 COMPETITIVE INHIBITION OF 5FU TRANSPORT BY URIDINE 92
4.11 EFFECT OF DIPYRIDAMOLE ON 5FU TRANSPORT 99
4.12 EFFECT OF NITROBENZYLTHIOINOSINE (NBTI) ON 5FU
TRANSPORT...................................................................... 103
4.13 EFFECT OF MODULATORS ON 5FU TRANSPORT...................... 104
4.13.1 ENHANCEMENT OF 5FU TRANSPORT BY MTX 104
5.0 DISCUSSION .................. 109
5.1 BIOLOGICAL MODELS........................................................................... 109
5.1.1 WALKER 256 ADENOCARCINOMA. ..................................... 109
5.1.2 SIGNIFICANCE OF THE ANIMAL MODELS......................... 110
5.1.3 SIGNIFICANCE OF THE CELL CULTURE SYSTEM 112
5.2 DETECTION OF PRODUCTS.................................................................. 117
5.2.1 1 9 F NMR SPECTROSCOPY. ...... 117
5.3 THE TRANSPORT OF 5FU INTO WALKER 256 TUMOR CELLS
AS AN ACTIVE TRANSPORT PROCESS ................................... 119
5.4 MECHANISM OF THE ACTIVE TRANSPORT PROCESS OF
5FUINTO WALKER256 CELLS............................................................. 132
v ii
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6.0 SUMMARY.. 140
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RE FE J E I ^ f I C 1 8 e e a s a s e e a e e s a e e o a e s s e o e e e a s e o a e e s e a e B a e a s a a o e a B B o s a e s s e t i s e e s e s e e B e 1 4 ’ 5
A l]P J P I H v l ^ T D I B o s a e e s e s a e e o e e o e e e s o e e s s o s o o s e e o o Q B e e a o a e s s e a B e f t a e a s e a a A e e s B e s e e s e o o e e s e B a 1 5 0
v iii
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LIST OF TABLES
TABLE
TABLE 4.1
TABLE 4.2
TABLE 4.3
TABLE 4.4
TABLE 4.5
• TABLE 4.6
TABLE 4.7
TABLE 4.8
TABLE 4.9
PAGE
FREE 5FU AND ITS METABOLITES IN THE TUMOR ASF OF
RATS, EXPRESSED AS RELATIVE UNITS (S/N).................... 58
5FU AND ITS METABOLITES IN THE LIVER ASF OF RATS,
EXPRESSED AS RELATIVE UNITS (S/N)......................... 61
EFFECT OF OUABAIN TREATMENT ON THE TRANSPORT OF
5FU INTO WALKER 256 TUMOR CELLS............................ 79
EFFECT OF EXTRACELLULAR GLUCOSE ON 5FU
TRANSPORT................................................................................ 81
EFFECT OF A GLYCOLYTIC INHIBITOR, IODOACETATE,
ON 5FU TRANSPORT ........................................................ 84
EFFECT OF EXTRACELLULAR pH ON 5FU TRANSPORT.. 85
EFFECT OF 2,4-DINITROPHENOL ON THE
TRANSPORT OF 5FU................................................................ 88
EFFECT OF STRUCTURAL ANALOGS ON THE TRANSPORT
O F5FU ........................... 91
EFFECT OF URIDINE ON THE RATE OF 5FU TRANSPORT
INTO WALKER 256TUMOR CELLS................. .94
ix
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TABLE 4.10 EFFECT OF DYPRIDAMOLE (DPD) ON THE TRANSPORT OF
5FU....................................................................................................... 100
TABLE 4.11 EFFECT OF NITROBENZYLTHIOINOSINE (NBTI) ON 5FU
TRANSPORT INTO WALKER 256 TUMOR CELLS .............. 103
TABLE 4.12 ENHANCEMENT OF 5FU TRANSPORT
BY METHOTREXATE.................................................................... 105
X
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LIST OF FIGURES
FIGURES
FIGURE 1.1
FIGURE 1.2
FIGURE 1.3
FIGURE 4.1
FIGURE 4.2
FIGURE 4.3
FIGURE 4.4
FIGURE 4.5
PAGE
STRUCTURE OF 5FU......................................................... 4
ANABOLISM AND CATABOLISM OF 5FU..................... 8
KINETICS OF TRANSPORT AS A FUNCTION OF
SUBSTRATE CONCENTRATION....................................... 23
A REPRESENTATIVE ,9 F SPECTRUM OF THE TUMOR
ASF FROM RATS 2 HOURS AFTER ADMINISTRATION
OF 150 mg/kg 5FU.............................................. 59
A REPRESENTATIVE ,9 F SPECTRUM OF LIVER ASF
FROM RATS 2 HOURS AFTER ADMINISTRATION OF
150mg/kg 5FU..................................................... 62
A REPRESENTATIVE I 9 F SPECTRUM OF FBAL IN
THE LIVER A SF....................................................................... 63
A REPRESENTATIVE ,9 F SPECTRUM OF THE ASF OF
CELLS AFTER EXPOSURE TO 1 mM 5FU FOR 1 -MIN 66
A REPRESENTATIVE 1 9 F SPECTRUM OF THE ASF OF
CELLS AFTER EXPOSURE TO 1 mM 5FU FOR 10-MI....67
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FIGURE 4.6 A REPRESENTATIVE 1 9 F SPECTRUM OF THE ASF OF
CELLS AFTER EXPOSURE TO 1 mM 5FU FOR 90-MIN... 68
FIGURE 4.7 ACCUMULATION OF FREE 5FU INTO THE WALKER 256
TUMOR CELLS AS A FUNCTION OF TIME...................... 69
FIGURE 4.8 RATIO OF INTRACELLULAR/EXTRACELLULAR 5FU 71
FIGURE 4.9 TIME COURSE OF 5FU TRANSPORT INTO WALKER 256
TUMOR CELLS IN THE PRESENCE AND ABSENCE OF
EXTRACELLULAR Na+ ....................................................... 74
FIGURE 4.10 A REPRESENTATIVE ,9 F SPECTRUM OF THE ASF OF CELLS
AFTER EXPOSURE TO 1 mM 5FU IN Na+ FREE BUFFER
FOR 1-MIN....................................... 75
FIGURE 4.11 A REPRESENTATIVE 1 9 F SPECTRUM OF THE ASF OF CELLS
AFTER EXPOSURE TO 5FU IN Na+ FREE BUFFER
FOR 5-MINS.......................... 76
FIGURE 4.12 RATIO OF INTRACELLULAR/EXTRACELLULAR 5FU IN
THE ABSENCE OF EXTRACELLULAR Na+ ...............................77
FIGURE 4.13 EFFECT OF EXTRACELLULAR GLUCOSE ON 5FU
TRANSPORT............................................... 82
FIGURE 4.14 EFFECT OF PRE-INCUBATION OF CELLS IN GLUCOSE FREE
BUFFER ON THE TRANSPORT OF 5FU..................................... 83
FIGURE 4.15 EFFECT OF EXTRACELLULAR pH ON 5FU TRANSPORT.. ..86
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FIGURE 4.16 A REPRESENTATIVE 1 9 F SPECTRUM OF THE ASF OF
CELLS AFTER 10-MIN WITH 5FU IN EXTRACELLULAR
pH 6.7.... 87
FIGURE 4.17
FIGURE 4.18
FIGURE 4.19
FIGURE 4.20
FIGURE 4.21
FIGURE 4.22
FIGURE 4.23
FIGURE 4.24
FIGURE 4.25
5FU UPTAKE AS A FUNCTION OF ITS CONCENTRATION 89
EFFECT OF URIDINE ON THE RATE OF 5FU TRANSPORT
INTO WALKER 256 TUMOR CELLS ............................... 95
DIXON PLOT OF THE COMPETITIVE INHIBITION OF 5FU
TRANSPORT BY URIDINE....................................... 97
CORNISH-BOWDEN PLOT OF COMPETITIVE INHIBITION
OF 5FU TRANSPORT BY URIDINE...............................................98
RECIPROCALS OF THE INITIAL RATES OF 5FU TRANSPORT
AGAINST DIFFERENT CONCENTRATIONS OF
DYPRIDAMOLE..................................................... ...101
A REPRESENTATIVE 1 9 F SPECTRUM OF THE ASF OF CELLS
EXPOSED TO 1 mM 5FU IN THE PRESENCE OF 20 mM
DYPRIDAMOLE...................................................... 102
EFFECT OF MTX ON THE TRANSPORT OF 5FU INTO
WALKER256 CELLS.................................. 106
A REPRESENTATIVE 1 9 F SPECTRUM OF THE ASF OF CELLS
EXPOSED T0 5FU AFTER 20-MIN PRE-INCUBATION WITH
MTX ....... 107
A REPRESENTATIVE ,9 F SPECTRUM OF THE ASF OF CELLS
EXPOSED TO 5FU AFTER 60-MIN PRE-INCUBATION WITH
M TX............... 108
x iii
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FIGURE 5.1 EFFECT OF PRE-INCUBATION IN SERUM FREE BUFFER ON
THE TRANSPORT OF 5FU ................................................... 116
FIGURE 5.2 RATIO OF INTRACELLULAR/EXTRACELLULAR 5FU ASA
FUNCTION OF THE EXTRACELLULAR pH............................ 128
xiv
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ACRONYMS
5FU, 5-fluorouracil;
FURD, 5-fluorouridine;
FUMP, 5-fluorouridine-5 ’-monophosphate;
FUDP, 5-fluorouridine-5 ’-diphosphate;
FUTP, 5-fluorouridine-5 ’-triphosphate;
FdUrd, 5-fluoro-2 ’ -deoxyuridine;
FdUMP, 5-fluoro-2 ’ -deoxyuridine-5 ’ -monophosphate;
FdUDP, 5 -fluoro-2 ’ -deoxyuridine-5 ’-diphosphate;
dFUTP, 5-fluoro-2 ’ -deoxyuridine-5 ’-triphosphate;
dUMP, 2 ’ -deoxyuridine-5 ’ -monophosphate
dTMP, thymidine-5 ’-monophosphate
dTDP, thymidine-5 ’ -diphosphate
dTTP, thymidine-5 ’-diphosphate
dHFU, 5-6 dihydro 5-fluorouracil
FUPA, a-fluoro-p-ureidopropionic acid;
FBAL, a-fluoro-P-alanine (2-fluoro-3 amino propionic acid);
MTX, methotrexate (4-amino-10-methylfolic acid)
NBTI, nitrobenzylthioinosine, 6-[(4-nitrobenzyl)thio]-9-p-D-
ribofuranosylpurine;
XV
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es, nitrobenzylthioinosine equilibrative sensitive;
ei, nitrobenzylthioinosine equilibrative insensitive;
DPD, dipyridamole;
ASF, acid soluble fraction;
DNP; 2,4-dinitrophenol;
IFN-a, interferon-a;
LV, leucovorin;
TMTX, trimetrexate;
PRPP, phosphoribosyl pyrophosphate;
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CHAPTER I
1.0 INTRODUCTION
Cancer treatment has been hampered in many cases by the inability of drugs to get to
their target site (s) in the right amount and at the right rate. For most anticancer
agents, a large percentage of the administered dose is degraded and eliminated before
it reaches the target site. For any drug however, to elicit its desired effect, a sufficient
amount has to be present at the target site (s) for a sufficient length of time.
Unfortunately, for many cancer drugs, it has been a great challenge to achieve a
sufficiently high concentration of the drug at the tumor site for a sufficient length of
time while minimizing uptake into normal tissues.
Solid tumors have varying degrees of perfusion. Most show an external layer of
proliferating cells, an intermediate layer of quiescent cells and an internal layer of
necrotic cells. These characteristics make them very poorly perfused and resistant to
the penetration of chemotherapeutic agents for cytotoxic action. Since however, the
effectiveness of most anticancer regimens have been correlated to the degree of
uptake, retention and intracellular activation to anabolites (1-5), it becomes necessary
to elucidate the mechanism by which the drug is transported into the tumor cells and
retained within the tumor cells. This information could be helpful in the
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manipulation of the transport process for maximum uptake into the tumor cells while
minimizing uptake into normal tissues. A good understanding of the transport
mechanism and certain characteristics of the transporter (s) may also allow the
identification of appropriate transport modulators that could be used to selectively
enhance transport into the tumor.
Nuclear Magnetic Resonance Spectroscopy (NMRS) has shown potential as a tool
that can be used for non-invasive monitoring of drugs and their metabolites in
various tissues of living organisms (1-3), especially at the target site. It involves
minimal perturbation of the system under study and allows real time data acquisition.
1 9 F-NMR spectroscopy has been used extensively to study directly the
pharmacokinetics and metabolism of fluorinated drugs at the target site (1-3, 7-9).
Though inherently poor in sensitivity when compared to other more conventional
techniques such as the use of radiolabeled drugs or HPLC, 1 9 F-NMR provides
exquisite chemical information. Hence, it allows the clear identification and
quantitation of all fluorinated compounds present in the tumor and other tissues of
interest provided they are present in a sufficiently high concentration.
We present here studies done in both animal model and cell cultures to understand
the mechanism of transport and regulation of the transport of 5-Fluorouracil (5FU),
an antineoplastic agent, into tumor cells. Although 5FU has been used as anticancer
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agent for more than 40 years and has been the subject of thousands of papers, the
mechanism of its transport into tumor cells is basically unknown. We also present
studies aimed at unveiling the nature and specificity of the transporter (s) involved in
the transport process. We have also initiated studies aimed at the identification of
effective transport modulators to selectively enhance the intracellular uptake of 5FU
into the tumor cells. These studies have been carried out using 1 9 F NMR
spectroscopy as the mode of analysis.
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1.1 5-FLUOROURACIL
1.1.1 WHAT IS 5FU?
5FU is a pyrimidine nucleobase belonging to the group of anticancer agents called
antimetabolites. It is a structural analog of the naturally occurring uracil, a precursor
of DNA synthesis. It is primarily used in the treatment of patients with solid tumors,
especially carcinomas of the breast, colorectal and gastrointestinal tract (10, 11). It
differs from uracil by the presence of a fluorine atom at the C-5 position. The
rationale for its synthesis was the enhanced utilization of uracil as the precursor of
RNA and DNA pyrimidines in a number of cancer cell lines. Hence, a slight
modification of the uracil molecule resulted in a potent antimetabolite.
Figure 1.1: Structure of 5FU
4
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1.1.2 CLINICAL USE OF 5-FLUOROURACIL
Since its synthesis in 1957 by Heidelberger et al (12), 5FU remains the most
extensively used chemotherapeutic agent in the treatment of colorectal and breast
carcinomas. While as a single agent 5FU produces objective tumor regression in
about 20 to 30% of treated patients, its major use today is as part of a combination
regimen.
5FU shows a rapid elimination from the blood with a half-life of about 10 - 20 min
(13) while human tumors have shown a prolonged retention of high concentrations of
free 5FU (2, 13). Pharmacokinetics of a single dose of 5FU as an intravenous (IV)
bolus injection in doses ranging from 300 and 600 mg/m2 show a rapid distribution
over a large volume and rapid elimination by the liver and kidney (11). The liver has
been demonstrated as having the highest level of the enzyme, dihydrouracil
dehydrogenase (EC 1.3.1.2) (14), required for the initial catabolism of 5FU. The
kidney, in which the activity of this enzyme is also high, contributes to elimination
by both degradation and active renal excretion (15), about 20% of 5FU being
excreted as the parent drug. The lungs have also been reported to be a major site of
5FU clearance (16).
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1.1.3 METABOLISM OF 5-FLUOROURACIL
Upon systemic administration, 5FU undergoes extensive metabolism. The
metabolism of 5FU follows two pathways: the anabolic pathway into active
anabolites, its fluoronucleotides and fluoronucleosides, and the catabolic pathway
into inactive catabolites. The anabolism of 5FU occurs primarily in the tumor while
the catabolism takes place primarily in the liver (11). Figure 1.2 summarized the
anabolism and catabolism of 5FU.
1.1.3.1 THE ANABOLIC PATHWAY
The anabolism of 5FU to fluoronucleotides such as fluorouridine 5’-triphosphate
(FUTP) and 5-fluoro-2 ’ -deoxyuridine-5 ’monophosphate (FdUMP), which takes
place primarily in the tumor, are necessary for its cytotoxic action (17,18). Several
enzymes are required for the conversion of 5FU to its fluoronucleotides. The extent
of tumor growth inhibition by 5FU has been correlated with the activity of one or
more enzymes catalyzing the initial metabolism of 5FU. For some cell lines, the
enzyme orotate phosphoribosyl-transferase (OPRT) (EC 2.4.2.10) has been shown to
play a major role in the initial metabolism, whereas for other cells uridine
phosphorylase (EC 2.4.2.3) is more important (19). It has also been shown that
nucleotides formed via the direct pathway (via OPRT) and the indirect pathway (via
FUR) are incorporated into different RNA fractions (20).
6
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1.1.3.2 THE CATABOLIC PATHWAY
5FU degradation takes place to some extent in all tissues, including tumor tissues,
which have been shown to contain very small amounts of the initial catalyzing
enzymes required for 5FU degradation (21). However, it is most significant in the
liver, which has the highest activity of the enzyme dihydrouracil dehydrogenase
(DPD) (EC 1.3.1.2) responsible for the initial degradation of 5FU by the reduction of
the pyrimidine ring and conversion of 5FU to dihydrofluorouracil (DHFU). This
enzyme then catalyzes the ring opening to form a-fluoroureidopropionic acid
(FUPA) which is then irreversibly converted by (3-alanine synthase (EC 4.2.99.13) to
£luoro-(3-alanine (FBAL), represented as the major catabolite excreted.
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Dihydrofluorouracil
(DHFU)
COOH
CHF
pHg '
NH
CO
N H g
COOH
CHF
c h 2
NH0
Catabolites
Fluoro-beta-alanin
(FBAL)
Fluoro-ureido-propionic aci
(FUPA)
I
Anabolites
5-Fluorouridin
(FUR)
5-Fluorouracil (5-FU
^ FUM
5-Fluorodeoxyuridin
(FdUR)
FdUM
FUD
FdUM -----^ FdUD
Thymidylate
Synthase
Figure 1.2: Anabolism and Catabolism of 5FU
FUTP
f
RNA
FdUT
I
DMA
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1.1.4 MECHANISMS OF ACTION OF 5FU
Several pathways have been postulated to account for the action of 5FU in tumor cells.
Of these, 3 major pathways have been largely agreed upon as the mechanisms of action
of 5FU once transported into the tumor cells.
These are
(i) Inhibition of thymidylate synthase by 5 -fluoro-2 ’ -deoxyuridine 5 ’ -monophosphate
(FdUMP). Thymidylate synthase catalyzes the central reaction in the de novo
synthesis of thymine nucleosides and nucleotides which is the rate limiting step in
DNA synthesis It is inhibited by formation of a tight and stable ternary complex with
FdUMP and 5,10-CH2 -tetrahydrofolate (22), which triggers their depletion. The
resultant shortage of thymine DNA precursors, the cessation of DNA synthesis and
the simultaneously ongoing RNA and protein synthesis are viewed as unbalanced
growth and as the basis for cell kill, termed thymineless death.
(ii) Incorporation of FUTP into all classes of RNA in tumor cells, mainly into nuclear
RNA (23) instead of uracil. The incorporation of 5FU into RNA via fluorouridine
triphosphate catalyzed by RNA polymerase interferes with the maturation of nuclear
RNA and subsequent inhibition of tumor cell growth.
9
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(iii) Incorporation of FdUTP into DNA, instead of deoxythymidine triphosphate
(dTTP), the normal substrate of DNA polymerase, and subsequent inhibition of chain
elongation (24).
1.1.5 MODULATION OF THE ACTION OF 5FU
5FU remains the agent of choice for the treatment of advanced colorectal cancer (10,
25-27). When used as a single agent, it produces objective tumor regression in about
of 20-30% of treated patients (25). Hence, a lot of research has been focused on the
biomodulation of 5FU in an attempt to improve its cytotoxicity and therapeutic
effectiveness. Several attempts to enhance the efficacy of 5FU have focused on the
alteration of tumor-cell metabolism to produce selective enhancement of cytotoxicity
(28). Modulation of 5FU by methotrexate (MTX) (11, 25), trimetrexate (TMTX),
interferon-a (IFN-a), leucovorin (LV), or N-(phosphonacetyl)-L-asparte acid
(PALA) (25), in addition to other combination chemotherapy with 5FU have
produced higher response rates than those with 5FU alone.
MTX has been shown to enhance 5FU-mediated cytotoxicity if administered several
hours before 5FU administration (29-32). It has been proposed that when MTX is
administered before 5FU, it inhibits de novo purine synthesis, resulting in an increase
in the intracellular concentration of phosphoribosyl pyrophosphate (PRPP), a co
substrate for the conversion of 5FU into FUMP and subsequently into FUTP.
10
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This yields a higher concentration of FUTP (33, 34) for subsequent incorporation
into RNA. Hence, the presence of MTX in the cells hours before the administration
and intracellular uptake of 5FU enhances the activity of 5FU. On the other hand,
MTX may also potentially interfere with the cytotoxic activity of 5FU by reducing
the intracellular pools of reduced folates necessary for the formation of the ternary
complex consisting of FdUMP,5,10-MeTHF, and TS (35).
In vivo inhibition of 5FU catabolism has also been exploited as a means to increase
the cytotoxic action of 5FU by increasing the availability of 5FU to tumors.
Administration of 5FU in conjunction with thymidine to block 5FU catabolism did
not show any clinical improvement over 5FU alone (36). Similarly, studies in rat
where the catabolism of 5FU was inhibited with thymidine showed an increase in
toxicity without a concomitant increase in observed clinical effect (37). Thus,
inhibition of 5FU degradation may not necessarily increase its therapeutic efficacy,
since toxicity increased as much or even more than antitumor activity, in these
studies. In another study, 5FU catabolism have been inhibited by the injection of 5-
diazouracil 2 hours before 5FU administration, resulting in a 75 and 66% decrease in
catabolites measured in the liver and kidney respectively (38). Inhibition of 5FU
catabolism by eniluracil, a potent inactivator of dihydropyrimidine dehydrohenase,
enhanced the localization of 5FU measured in the tumor (39).
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1.2 TRANSPORT PROCESSES
1.2.1 TRANSPORT OF 5-FLUOROURACIL
Why is an understanding of 5FU transport important? Prior studies have documented
that patient response to chemotherapeutic treatment with 5FU is correlated with the
degree of transport, uptake and trapping of free 5FU in the tumor (1-5). Studies by
Wolf, Presant et al (1994), had shown that patients’ response to chemotherapy with
5FU was strongly associated (p<0.00001) with a tumoral t1 /2 of 5FU of 20 minutes or
longer (3). Hence, “trapping” of free 5FU in the tumor has been documented as a
necessary condition to observe cytotoxic effect (1, 2). Therefore, the need to fully
understand the mode and mechanism of transport of 5-Fluorouracil into tumor cells.
This understanding might allow effective manipulation of the transport process to
optimize uptake into the tumor while minimizing uptake into normal tissues.
There are three major organs where transport of 5FU occurs. These are the intestinal
tract, the liver hepatic pathway and transport into the tumor. In the intestinal
pathway, the transport of 5FU is across the intestinal tissue from the apical to the
basolateral side. In this pathway, transport into the cell is not intended, although,
there could be leakage into the cell. In the liver, the uptake of 5FU is of a degradative
nature and is metabolically driven. When 5FU degradation in the liver is blocked,
there is a complete efflux of the 5FU transported into the liver. There is no trapping
12
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of free 5FU in the liver. Lastly, in the tumor the transport of 5FU into the cells
appears to be regulated by membrane transporters. These transporters regulate the
influx and possibly the efflux of 5FU in tumor cells. It does not appear to be
metabolically driven as initially thought, as the presence of free 5FU has been
observed in tumors long after its clearance from the plasma (13).
Most of the studies directed at understanding the mode of transport of 5FU have
focused on the transport across the rat small intestinal tissues. In this model, the
transport of 5FU has been shown to proceed by both a saturable and a non-saturable
process (40-43). At low concentrations, active transport is the predominant mode of
uptake while passive diffusion prevails at high concentrations of 5FU (40-43).
The active transport of 5FU across the rat small intestine has also been shown to be a
Na+ dependent process. 5FU uptake into tissue rings cut from rat small intestine
show a Na+ dependent active transport process at low substrate concentrations with
the active transport process significantly inhibited by the Na+ /K+ -ATPase inhibitor,
ouabain (40,41) and by replacement of Na+ with choline in the incubation medium
(40, 41). The active transport of 5FU in this model was also significantly inhibited by
structurally related analogs such as uracil and thymine but not by cytosine. The mode
of uptake of cytosine has been observed to be by passive diffusion (44).
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Also, in the intestinal everted sacs, the initial uptake of 5FU was found to be Na+
dependent, with inhibition of uptake when Na+ in the medium was replaced by
K+ (45). Passive transport was significant at higher concentrations. The active
transport was inhibited by structurally related pyrimidines (uracil and thymine) (42,
45) and by 2, 4-dinitrophenol (DNP), a metabolic inhibitor but not by purines
(adenine and guanine) and pyrimidine nucleosides (thymidine and uridine) (45). On
the other hand, studies in the brush border membrane vesicles (BBMVs) of rats have
documented a Na+ independent uptake of 5FU, which was slightly inhibited by uracil
but not by thymine (45). Also in the rat liver, active transport of 5FU has been shown
to exist, with this process significantly inhibited by 2,4-dinitrophenol (46).
In the human erythrocytes, the uptake of 5FU is by a non-facilitated diffusion
process (47,48). The transport into human erythrocytes was partially inhibited by
adenine, hypoxanthine, thymine, and uracil but was insensitive to inhibition by
nucleosides or inhibitors of nucleoside transport.
In addition to the above studies, a few studies on the transport of 5FU have been
carried out in tumor cells. These include studies in Novikoff hepatoma cells, Ehrlich
Ascites tumor cells and Lettre cells (49, 50, 51). In the Novikoff hepatoma cells, the
transport of 5FU has been postulated to occur by a facilitated diffusion process (49)
while in the Ehrlich Ascites tumor cells, an active transport mechanism has been
documented (50). The transport of 5FU into the Ehrlich Ascites tumor cells was
14
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competitively inhibited by pyrimidine analogs as well as by glycolytic inhibitors.
The active transport of 5FU in these cells was directly coupled to cellular ATP and
independent of Na+ , with intracellular accumulation of 5FU occurring even in the
absence of extracellular Na+ (50). Studies in the Lettre cells have also documented a
Na+ independent transport of 5FU and the dependence of transport on the tumor pH
gradient (51).
The pH gradient across tumor cell membranes has been suggested to affect the
transport and trapping of free 5FU in tumors. In general, solid tumors in vivo have a
negative pH gradient, the reverse of normal tissues. The intracellular pH is alkaline
while the extracellular pH is acidic, mostly because of the high glycolytic rate
associated with tumors and subsequent extrusion of cellular acids. Ojugo et al (1998)
reported in Lettre that an increase in the negative pH gradient (- pH) across the tumor
cell membrane correlated with an increase in the uptake of 5FU (5FUi n t /5FUe x t ratio)
by the cells (51). In another study, Guerquin-Kem et al (1991) showed that the half-
life of elimination of 5FU from rat fibrosarcoma was 2.5 fold longer at pHi < 6.9
than at pHi 7.3 (52). Could the pH gradient across tumor cell membranes be the
mechanism by which 5FU is transported and free 5FU trapped within the tumors?
Are there other mechanisms that regulate the transport and intracellular trapping of
free 5FU in the tumor cells?
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1.2.2 NUCLEOBASE TRANSPORT SYSTEM
The nucleobase transport system has not been as extensively studied as the transport
system for nucleoside uptake into mammalian cells. Among the nucleobases, most of
the attention has been focused on the purine nucleobases such as hypoxanthine, with
much less attention given to the study of the pyrimidine nucleobase transport system.
Hence, much of this discussion is focused on what is known about the purine
nucleobase transport system in mammalian cells.
A number of distinct carrier-mediated transport processes have been proposed to
account for the permeation of purine nucleobases across mammalian plasma
membranes. Like the nucleoside transport system, two major classes of nucleobase
transporters, equilibrative and concentrative nucleobase transport systems, have been
demonstrated to occur in mammalian cells. The equilibrative transporters are present
in a number of cell types and have been more predominantly characterized in the
human erythrocytes (53, 54). They show broad selectivity for both purine and
pyrimidine nucleobases. In addition to the equlibrative nucleobase transporters,
concentrative Na+ dependent nucleobase transporters have been observed in a
number of cell lines. These include LLC-PK, cells (55, 56), brush border membrane
vesicles from guinea-pig kidney (57), rat intestine (40, 41, 45, 58), choroid plexus
(59) and OK cells from the late proximal tubule of the opossum kidney (60). These
transporters are proposed to mediate the translocation of specific nucleobases.
16
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A sodium-dependent active nucleobase transport system has also been demonstrated
in epithelial tissues. In the everted rat jejunum, rat jejunal tissue rings, isolated rat
jejunal loops and vascularly perfused rat jejunum, the mucosal to serosal transmural
transfer of uracil, 5-fluorouracil and thymine at concentrations < 200 pM was
reported to occur against a concentration gradient (40, 58, 61). The uptake of 5FU in
the rat jejunal tissue rings exhibited a saturable uptake process (Km 74 pM) and was
abolished in the absence of Na+ (62). Inhibition studies of the mucosal to serosal
transfer across these models show a broad substrate specificity of this Na+ dependent
nucleobase transporter, accepting both naturally occurring purine and pyrimidine and
several analogues (40, 58, 62).
The Na+ dependent transport system for nucleobases has also been demonstrated in
cultured animal cells. Reported in renal epithelial cell line, LLC-PK, is the presence
of a high-affmity Na+ nucleobase cotransporter (56), which is very similar to the
previously described Na+ -dependent system in rat small intestine. Studies on
hypoxanthine uptake in this model show an accumulation against a concentration
gradient in the presence of Na+ with a saturable influx (Km 1 pM at 22°C) (56). The
transport was inhibited by several analogues such as uracil, 5FU, thymine and
guanine. Nucleoside transport inhibitors were observed to be potent inhibitors of the
Na+ dependent hypoxanthine influx whereas, interestingly, nucleosides had no effect
on the transport process.
17
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Similarly, in both the brush border membrane vesicles from guinea pig kidney and
OK cells, a renal epithelial cell derived from the late proximal tubule of the opossum
kidney, and in the rabbit choroid plexus, there is evidence for the presence of a Na+
dependent nucleobase transporter (57, 59, 60). The Na+ stimulated hypoxanthine
uptake in the rabbit choroid plexus was inhibited by naturally occurring purine and
pyrimidine nucleobases as well as by the nucleoside analog, dideoxyadenosine (59).
The human erythrocyte, in which the transport of nucleobases has been extensively
studied, the transport of hypoxanthine and adenine are shown to occur by a single
saturable system independent of the nucleoside carrier (63). The order of affinity of
natural occurring nucleobases in the human erythrocyte has been determined as
adenine > guanine > hypoxanthine > uracil (Km values of about 0.013,0.037, 0.18
and 5.8 mM respectively at 37°C) (47, 64). In addition, cultured mammalian cells
such as human T-lymphoblastoid CCRF-CEM cells and pig renal epithelial LLC-PKj
also show similar high-affinity, broad specificity nucleobase carrier (57, 65-67).
Some studies on cultured mammalian cells have reported both the equilibrative-
sensitive (as) and the equilibrative-insensitive (ei) nucleoside transporters to play a
role in the translocation of nucleobases. Involvement of the es nucleoside transporter
was found in S49 cells, which lack the purine nucleobase carrier but expresses
mainly the es nucleoside transporter. Hypoxanthine was transported in these cells
with a saturable influx, though slower than that observed in cells with specific purine
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nucleobase carrier (Km 0.8 - 1 mM) and the influx was inhibited by thymidine (68,
69).
The ei nucleoside transporter has also been implicated in the transport of nucleobases
in HTC rat hepatoma, CHO, Chinese hamster lung and Ehrlich Ascites tumor cells
(63). Hypoxanthine influx in these cells was inhibited by uridine and other
nucleosides while nitrobenzylthioinosine (NBMPR) failed to inhibit the uptake (44,
66, 68, 70, 71). Also, in the Novikoff hepatoma cells, which express a large
percentage of ei nucleoside transporters, hypoxanthine exhibited a very high affinity
for the nucleoside transporter (Km 300-400 uM) (72).
1.2.3 NUCLEOSIDE TRANSPORT SYSTEM
Physiological nucleosides and nucleobases and most nucleoside analogues are
hydrophilic in nature and require protein transporters for their movement into and out
of cells. Therefore, the presence or absence of nucleoside and nucleobase transporters
on cells affect the pharmacokinetics, disposition and in vivo biological activity of
physiologically occurring compounds, as well as nucleoside and nucleobase drugs.
Hence, a lot of effort has been focused towards the characterization of the nucleoside
transport system in various mammalian cells. Studies on the nucleoside transport
system show that the permeation of nucleosides across the plasma membrane of
19
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mammalian cells is very complex and is mediated by multiple transport proteins.
These transporters fall into two basic categories: (1) the equilibrative (facilitated
diffusion) carriers that mediate both the influx and efflux of nucleosides and (2) the
concentrative, Na+ -dependent carriers, which mediate only the influx of nucleosides
under physiological conditions (73).
The equilibrative nucleoside transport processes (facilitated diffusion) are further
classified into two distinct forms based on their sensitivity to inhibition by
nitrobenzylthioinosine (NBTI) (74-77). One form termed the es (equilibrative
sensitive) is strongly inhibited by nanomolar concentrations of NBTI, as a result of
binding ofNBTI to high-affinity binding sites on the plasma membrane. The second
form, designated ei (equilibrative insensitive) is not associated with such high
affinity binding sites, hence is resistant to inhibition by nano-molar concentrations of
NBTI and only inhibited by micromolar concentrations ofNBTI (75-77).
Mammalian erythrocytes and some cultured cell lines express mainly the es
nucleoside transport (78-81) whereas some cell lines such as the Novikoff hepatoma
cells and the Walker 256 carcinosarcoma cells lack high affinity NBTI binding sites
(74, 76). These cells express only the ei nucleoside transporter. However, most cells
express both forms of nucleoside transport in varying proportions (75-77, 82).
20
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Kinetically, both forms exhibit broad specificity, but the substrate affinities, turnover
numbers and carrier mobility show considerable heterogeneity (44, 83).
In addition to the non-concentrative facilitated transport system for nucleoside
transport, active (concentrative) Na+ dependent nucleoside transporters have been
reported in a variety of experimental preparations, including perfused tissues,
isolated intact cells and membrane vesicles (for review see 61). In the rabbit choroid
plexus, uridine and thymidine accumulated in the unmodified form to over 10 times
the extracellular concentration and the saturable uptake (apparent Km values of 7.2
andl3.2 pM for uridine and thymidine, respectively) was inhibited by dinitrophenol
and iodoacetate (84-86). Inhibitors of non-concentrative, facilitated nucleoside
transport, such as NBTI, dipyridamole and diazepam, did not inhibit the Na+
dependent nucleoside transport but were found to enhance the accumulation of
uridine (44, 84-86). This perhaps, occurs by inhibition of uridine efflux through the
non-concentrative transporter.
Similarly, Na+ dependent active nucleoside transport systems have been detected in
the brush-border membrane vesicles (BBMVs) of rat kidney and of rabbit intestine,
in isolated guinea pig intestinal epithelial cells and in mouse spleen cells (87, 88)
exhibiting broad substrate specificity. In the BBMVs of rat kidney, nucleosides
accumulated in the vesicles to a level 20-times the extracellular concentration within
21
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20-40 sec of incubation at 37°C in the presence of 100 mM NaSCN whereas only
equilibrium was reached in the presence of 100 mM KSCN (87, 88).
1.2.4 MODULATION OF 5FU TRANSPORT BY METHOTREXATE (MTX)
MTX is one of the agents that have been used to modulate the activity of 5FU by
affecting its anabolism to active fluoronucleotides and fluronucleosides when
administered before 5FU is given. It has been shown that when MTX is administered
about 4 hours before the administration of 5FU, it enhances the anabolism of 5FU
(31, 32) by increasing the levels of 5-phosphoribosylpyrophosphate (PRPP) in the
tumor. This enzyme catalyzes the formation of FUMP and FUTP from 5FU for
subsequent incorporation into RNA.
In addition to MTX alteration of tumor cell metabolism of 5FU, studies by El-
Tahtawy and Wolf (1991) showed a significant decrease (estimated at more than 3
orders of magnitude) in the elimination rate constant of 5FU when rats bearing the
Walker 256 adenocarcinoma were pre-dosed with MTX (31). This, compared to the
rate constant for the conversion of 5FU to fluorinated nucleosides and nucleotides,
which increased by 2.5 fold (31), suggest that MTX may be affecting the transport of
5FU into the tumor cells as well. In more recent clinical studies by Presant and Wolf
et al, about a 42% increase in the intratumoral tm of 5FU was observed when patients
were administered intravenous MTX before 5FU was given (89).
22
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1.2.5 KINETICS OF A SATURABLE TRANSPORT
1.2.5.1 MICHAELIS -MENTEN EQUATION
Mediated transport across cell biomembranes is usually related to the substrate
concentration (raised to the power of the number of like substrate ions or molecules
transported together) by a simple rectangular hyperbola (Fig. 1.3).
1 0 0 0
m ax
500 ~
0.2 0.3 0,4 o 0.1 0.5
[SJ, mM
Figure l.3 (90) Kinetics of Transport as a function of substrate concentration
The shape of the curve implies that the substrate binds transiently to a mediating
structure know as a transport protein, which is usually limited in supply. The
transport process can also be represented by a simple equation:
K, K2
S, + M < = > MS -» M + S2 (Equ. I)
I' m
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Where Sj and S2 represent the same substrate molecule on side 1 and side 2 of the
membrane respectively, M is the transport protein, K, is the rate constant for the
formation of MS, K, is the rate constant for the dissociation of MS back to S, and M
and K2 represent the rate constants for the translocation of S to side 2 of the
membrane and its dissociation from M.
When the concentration of S on side 1 is sufficiently high, M will become almost
completely occupied or saturated (i.e., it will be almost entirely MS), and the
transport process will approach a maximum velocity, Vmax. The concentration of S
at one-half maximal velocity is Km. The Vmax and Km values are a consequence of
the close biophysical interactions between small ions and molecules and the protein
molecules that catalyze their biomembrane transport.
1.3 ASSAY METHODS FOR STUDYING THE TRANSPORT OF 5FU
1.3.1 ANIMAL STUDIES
The assay methods for studying the transport of 5FU across tumor membranes using
animal models are limited. Female Sprague Dawley rats bearing the Walker 256
adenocarcinoma have used as model to monitor the appearance and elimination of
5FU using 1 9 F NMR Spectroscopy (31). While the animal models would more
closely mimic the conditions in humans, particularly the impact of the interstitial
fluid space on the transfer of 5FU into the tumor, the structural complexity would not
2 4
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allow the adequate manipulation of the environment to allow the study of one
process at a time. Hence, most transport studies have been carried out in cultured
cells.
1.3.2 CELL CULTURE STUDIES
The assay methods for studying the transport of substances across cell membranes
using cell cultures are very vast. Refs 42 and 61 provide good reviews on the
different types of cells and experimental preparations used to study the nucleoside
and nucleobase transport systems. The movement of many substances across cell
membranes is accounted for by their dissolution in the lipid phase and subsequent
diffusion through the lipid phase to the opposite side of the membrane. However,
there are substances that cannot be included in this simple model. These substances
require transport proteins that mediate their transfer from one side of the membrane
to the other, either down their electrochemical gradient or against the prevailing
electrochemical gradient.
In cell culture studies, radiolabelling of the substrate (e.g. 5FU) whose transport is
studied is often the method of choice. This technique is very sensitive and is able to
accurately measure the transfer of the substance from one side of the membrane to
the other within a few seconds. This method measures the total radioactivity present
on the opposite side of the membrane. However, this technique is not able to
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distinguish between the parent compound and its metabolites as it gives a record of
the sum of the parent compound and metabolites present. HPLC is another sensitive
method that has been used to measure the transport of 5FU into cell extracts. This
method allows the detection of 5FU in plasma or tumor tissues of up to 1 ng/ml.
However, the HPLC method requires the extraction of the tissue of interest and
extensive sample preparation.
I 9 F NMR spectroscopy is an alternative technique that can be used to study the
transport of 5FU across tumor cell membranes. This technique allows both an
invasive and a non-invasive study of the transport of 5FU. 1 9 F NMR spectroscopy
can and has been used to monitor directly the metabolism of 5FU in intact
multicellular tumor spheroids formed by human colon carcinoma cells HT-29 (91) in
a perfused system. Use of tumor spheroids formed from cultured cells permit the
study of drug transport in the intact cell without any extensive extractions using 1 9 F
NMR spectroscopy. Compared to the previously mentioned techniques, 1 9 F NMR
Spectroscopy has several advantages which includes
(1) The ability to study the transport of 5FU in the tumor cells or in any tissues of
interest without prior extraction and separation.
(2) No radiolabelled drugs are required since the fluorine atom is an integral part of
the drug.
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(3) The ability to differentiate between the fluorinated parent compound, as well as
the fluorinated metabolites.
1.3.3 NMR SPECTROSCOPY METHODS
The nuclear magnetic resonance (NMR) spectroscopy is a technique that is based on
the absorption of radiofrequency (RF) radiation by an atomic nucleus with odd mass
number such as *H , 1 3 C, 1 9 F, and 3,P. An NMR signal arises when a nucleus that has
been aligned in a magnetic field is subjected to electromagnetic radiation
(radiofrequency). The signal is due to a change in the nuclear spin state and occurs at
a characteristic frequency for a given nucleus. The NMR signal obtained is directly
proportional to the number of nuclei producing it (92).
The exploitation of the NMR phenomenon requires three basic components:
(1) Nuclei with magnetic moment (example, 'H, 1 3 C, or 1 9 F, but not 1 2 C).
(2) A relatively large and preferably homogenous magnetic field
(3) Tuned coil (s) to transmit energy at the resonance frequency of the nuclei
concerned (in the radiofrequency range) and to detect the resultant disturbances of
equilibrium.
The chemical shift (8) of a specific NMR nucleus is measured from a reference
signal and is independent of the magnetic field strength (po) but dependent upon its
chemical environment.
27
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The principal advantage of the NMR technique is the ability to non-invasively detect
and measure the real time metabolite levels without preliminary extraction or
chemical manipulation. Being non-invasive in nature, this technique allows repeated
in- vivo measurements of concentrations of selected molecules in normal and tumor
tissues on both humans and animals. This attribute makes it possible to monitor the
biodistribution, targeting, metabolism, and kinetics of the fluorinated drug in the
tissue or tumor of interest in clinical studies. Unlike conventional methods of
analysis such as radiolabelling which gives information only on the sum of all
compounds present, NMR spectroscopy provides detailed information on the nature
of the compound (s) present, provided that such compound (s) are present in near
micro-molar concentrations.
The NMR techniques however, are intrinsically insensitive because the thermal
difference between high and low nuclear spin states is small. The detection limit for
in vivo 1 9 F NMR spectroscopy (the easiest nucleus for pharmacokinetic studies) is
about 0.01 to 0.1 mmol. Also, restricted molecular motion (e.g. macromolecular
binding of a drug) may alter the relaxation times and make detection even more
difficult through severe broadening of spectral lines.
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1.3.3.1 1 9 F NMR SPECTROSCOPY
1 9 F NMR spectroscopy has been used extensively in the detection and analysis of
fluorinated compounds in biomedical research. Application of the 1 9 F NMR
technique requires a “built-in” fluorine atom in the nuclei or drug of interest or the
incorporation of fluorine with minimal perturbance of the drug’s properties. 1 9 F
nuclei have a spin of l A and have been referred to as the easiest nucleus for
pharmacokinetic studies. It has a high NMR sensitivity, 84% of the sensitivity of 'H,
a wide chemical shift range (about 200 ppm) and the added advantage of the absence
of natural fluorinated compounds in the human body (except some fluoride ion)
eliminating possible interfering background signals. This technique allows the non-
invasive, real time, and simultaneous detection and quantitation of specific
fluorinated drugs and all of its fluorinated metabolites in the tissue of interest. It does
not require any radiolabelled compounds since the target nucleus is an integral part
of the drug and its metabolites.
1.3.3.2 EXPERIMENTAL PROCEDURE AND PROBLEMS
The experimental method required to acquire an NMR spectrum is very
straightforward. This technique requires that the sample to be examined must be a
liquid or gas because solids give very broad signals causing much of the fine
structure to be lost. So if the sample under investigation is a solid, it is dissolved in
or mixed with suitable solvent, a small amount (< 5%) of reference compound added
2 9
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(or sometimes the solvent peak may be used as the reference) and the spectrum
obtained (92).
The standard size of the sample tube for most routine *H spectrometers is 5 mm OD
(outside diameter) and this requires about 0.4 ml of solution for normal operation.
For 1 3 C measurements, where sensitivity problems are such that much more sample is
required than for proton NMR, larger sample tube diameters are used. As large as 18
mm OD sample tubes are commercially available. However, when there is no need
for larger quantity of sample, a smaller size tube is preferred because both resolution
and signal-to-noise are better in the smaller tube. For 1 9 F measurements, 5 mm or 10
m m OD size tubes are used. The 10 mm OD size tubes require a 3.0 - 3.5 ml of
sample volume.
The amounts of sample required for all NMR measurements are high compared to
other analytical techniques and other spectroscopy techniques such as infrared and
ultraviolet because of the inherently low sensitivity of this technique. Before the
advent of routine Fourier Transform (FT) spectrometers with inherently greater
sensitivity, most routine proton spectrometers required a 10-15 mg of sample for a
reasonable spectrum. However, with the new routine FT spectrometers smaller
amounts of samples are required, usually 1-10 mg of sample for proton
spectrometers. For 1 3 C spectra, owing to its low sensitivity, the major requirement is
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to obtain as concentrated a solution as possible in order to obtain a reasonable
spectrum in as short a time as possible.
The case of sparingly soluble samples is a recurrent problem in NMR. It is very
important to obtain a clear mobile solution, as any solid particles, or a viscous
solution will seriously impair the resolution of the spectrum. The sample solubility,
the absence of any signals from the solvent occurring in the spectral region under
investigation determines the suitability of the solvent to be used. Deuterochloroform,
CDCI3 is the most commonly used solvent. It is mostly invisible in the proton
spectrum.
For NMR quantitation, the choice of a suitable reference compound is very
important. For ‘H and also for 1 3 C NMR, the recommended reference is
tetramethylsilane, Si(CH3 )4 , usually termed TMS. The nomenclature is the 5-scale,
which takes the TMS peak as 0 and increases in a downfield direction. This direction
is the direction of increasing frequency, therefore spectra are always measured from
right to left in frequency units (92). For 1 9 F NMR, 1,2-difluorobenzene and 5-Fluoro-
DL-tryptophan are commonly used reference compounds.
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1.3.3.3 EXPERIMENTAL MEASUREMENTS
1.3.3.3.1 CHEMICAL SHIFT MEASUREMENT
The chemical shift of a specific nucleus is the precise frequency of the NMR signal
and is dependent upon its chemical environment. The chemical shift (8) is usually
specified relative to a reference standard. It is expressed in terms of a dimensionless
unit of parts per million (ppm) and is independent of the magnetic field strength (po).
§ = (vs - V r ) / v r * 1 0 A6
where vs and vR are the resonance frequencies of the line of the sample and of the
reference respectively.
The reference standard could be internal, that is in the same tube as the sample under
study or it could be external (in a different capillary tube inserted into the tube
containing the sample under study).
1.3.3.3.2 SENSITIVITY
One major limitation of the NMR spectroscopy technique is the inherent lack of
sensitivity relative to other analytical techniques such as HPLC or the use of
radiolabelled materials. The main reason for this low sensitivity is the small
magnitude of the energy changes involved in NMR transitions. Therefore, the
sensitivity of the technique is exponentially proportional to the size of the energy
changes concerned. In general, the signal-to-noise (S/N) ratio has been used as a
measure of the NMR sensitivity.
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The signal-to-noise (S/N) ratio of a particular peak is dependent on (i) the nucleus of
interest and its gyromagnetic ratio, (ii) the size of the sample and the concentration of
the chemical species present, and (iii) the magnetic field strength of the spectrometer.
Several methods have been exploited in an attempt to enhance the sensitivity of the
NMR technique. Firstly, an increase in the strength of the magnet has been employed
as a means to increase sensitivi ty, since the energies of the various spin states depend
on the strength of the applied magnetic field.
Accumulation of several spectra from a sample is another means exploited to
enhance NMR sensitivity. Under this condition, the NMR signals will add
coherently, whereas the noise, being random, will only add as the square root of the
number of spectra accumulated. This leads to an overall improvement in the signal-
to-noise (S/N) ratio, which is the square root of the number of spectra accumulated.
The main limitation of this method however, is the length of time taken to obtain an
individual spectrum.
Paramagnetic relaxation by the addition of paramagnetic materials such as acetyl-
chromium acetate Cr(acac)3 to samples that posses long relaxation times have been
used to enhance the signal-to-noise ratio of a particular sample and increase the
NMR sensitivity.
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1.3.3.4 QUANTITATION OF AN NMR SPECTRUM
Quantitation of an NMR spectrum can be very challenging. Ideally, it would require
placing a reference sample of known concentration in the same field as the sample
under study under identical conditions so the ratio of the specific peak integrals or
intensity is used to estimate the concentration of substances in the extracts. However,
this is almost impossible and so several methods have been developed to allow the
quantitation of an NMR spectrum. These include:
(1) Chemical shift: In some cases, the chemical shift measurement has been used to
obtain some quantitative information. For instance, the number of S atoms in the
sulfur chain of bisdialkyl polysulphides determines the chemical shift of the CH2
(93).
(2) Peak Heights: Under certain conditions, such as if the line widths are identical,
the peak heights are proportional to the number of nuclei when they are determined
by the field homogeneity and not by T2 , the spin-lattice relaxation time. In some
instances, both area and peak height measurements have been used simultaneously to
quantitate an NMR spectrum.
(3) Area Measurements: Measurement of the area under the curve is the most
frequently used method for NMR quantitation. This can be done in several ways such
as by triangulation, by cutting and weighing or through electronic integration.
Quantitation by area measurements requires calibration of a known amount of the
compound under investigation. The developed calibration curve of ratio of peak areas
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against the concentration of the references is used to estimate the concentration of the
substance in the extracts.
(4) The signal-to-noise (S/N) ratio, in addition to the ratio of peak intensities has
been used for NMR quantitation. In this instance, the S/N ratio of a particular region
on the spectra is electronically or manually calculated and ratios of reference and
sample peak intensities are used to estimate the concentrations of substances in the
extracts. This method also requires the calibration of known concentrations of the
sample under study against the signal to noise ratio and ratios of peak intensities of
the sample and the reference standard. The developed calibration curve is used to
estimate the concentration of the substance in the extracts.
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CHAPTER II
2.0 STATEMENT OF OBJECTIVE
It has become increasingly important to understand the mechanism and regulation of
the transport of 5-Fluorouracil (5FU) into tumor cells. The nature and extent of 5FU
transport into tumor cells differ significantly from its transport into non-tumor
tissues. Despite the fact that there is an increased uptake of 5FU by tumor tissues
compared to non-tumor tissues, the cytotoxicity of 5FU has been hindered by the low
amount of drug that actually gets transported into the tumor cells. Less than 2% of
the administered dose of 5FU eventually gets transported into the tumor cells.
The cytotoxicity and the degree of effectiveness of 5FU has been correlated with its
degree of uptake, retention and intracellular activation (1-5) in tumors. Therefore, a
good understanding of the mechanism and the factors that regulate the transport of
5FU into tumor cells could allow the identification of appropriate transport
modulators that would enhance its uptake into tumor cells. The information so
obtained might allow a more rational combination therapy with 5FU.
The studies reported here were aimed at elucidating the mechanism of transport of
5FU into tumor cells, whether it is by an active transport process, by facilitated
diffusion or by simple diffusion across the tumor cell membranes. With the
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understanding of the mode of uptake, further studies were done to understand the
nature and specificity of the transporter (s) that mediate 5FU transport into tumor
cells.
These studies have been carried out in cell cultures of the Walker 256
adenocarcinoma cells and in rats bearing the same tumor model. The initial studies in
animals made it possible to evaluate the significance of the tumor interstitial fluid
space in the transport of 5FU into the tumor cells. While transport of 5FU from the
tumoral blood pool into the tumor interstitial fluid space is postulated to be largely
by passive diffusion, the transport from the tumor interstitial fluid space into the
tumor cells, appear to be regulated by membrane transporters. The animal model
however, was too complex and could not permit an adequate study of the factors that
regulate 5FU transport into the tumor cells. Therefore, more studies on the
mechanism and regulation of 5FU transport across tumor cell membranes were done
in cell cultures.
The isolated system of the cell cultures made it possible to control the various
processes going on and allowed the study of one process at a time. Some questions
addressed were; the rate of transfer of 5FU into tumors, the dependence of the
transport process on glucose and Na+, the competition between structural analogs,
what transporter (s) mediate 5FU transport into tumor cells and the identification of
agents that enhance or inhibit 5FU transport into tumor cells.
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Though these studies were done in ex-vivo and in-vitro systems, we believe that the
mechanism of transport of 5FU described herein, appears to be the general
mechanism of 5FU transport across tumor cell membranes in cell cultures, and could
also be the mechanism of transport of this agent in human tumors. The information
obtained from these studies could make it possible to maximize the utility of 5FU as
an anticancer agent by identifying appropriate modulators of its transport in
combination chemotherapy.
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CHAPTER III
3.0 MATERIALS AND METHODS
3.1 DRUGS AND CHEMICALS
The following products were used and their sources were:
5-Fluorouracil (5FU) - the clinical preparation that was used, was the sodium salt in
water at a concentration of 500 mg/10 ml (Roche Laboratories, a division of
Hoffmann-La Roche Inc., Nutley, NJ). Uracil, thymine, cytosine, uridine, cytidine,
dinitrophenol, dipyridamole, ouabain, nitrobenzylthioinosine, methotrexate and
deuteriumoxide were purchased from Sigma Aldrich (Aldrich Chemical Co.,
Milwaukee, WI). RPMI medium 1640, fetal calf serum, Medium 199, horse serum,
trypsin-EDTA and all other materials for cell culture were purchased from Gibco
Laboratories, Life Technologies, Inc. (Grand Island, NY 14072). Ketaset [Ketamine
Hydrochloride] 100 mg/ml was purchased from Veterinary Products, Bristol
laboratories (Bristol-Myers Co., Syracuse, NY 13201). Rompun [xylazine] 20 mg/ml
was purchased from Haver Mobay Corporation (Shawnee, Kansas 66201). All other
chemicals used were of the highest commercially available grade. NMR tubes (10-
mm OD) were purchased from Wilmad glass (Wilmad/Lab Glass, Buena, NJ).
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3.2 WALKER 256 SOLID CARCINOMA FOR ANIMAL STUDIES
The Walker 256 tumor cell line was donated to our laboratory by Dr. T. Khwaja,
Cancer Research laboratory, USC, and has been frozen in liquid nitrogen since 1978.
It was reactivated in 1995.
3.2.1 TUMOR TRANSPLANTATION
Female Sprague Dawley rats weighing about 200 g and about 6 -8 weeks old were
anaesthetized with a 1:1 mixture of Ketamine (100 mg/ml) and xylazine (Rompum,
20 mg/ml). Frozen cells were thawed quickly at 37°C and centrifuged at 4000 rpm
for 10-mins and the freezing medium decanted. The cells were re-suspended in
saline, centrifuged at 4000 rpm for 10-mins and the saline decanted. The viability of
the cells was routinely determined by using the trypan blue exclusion test. The
washed cells were re-suspended in 1 volume RPMI 1640 medium and were injected
subcutaneously (1 x 107 cells) into the right or left thigh of the rats. Animals were
observed for tumor development and growth. Tumors were allowed to grow to a size
of about 1 - 1.5 cm, which usually took about 7-10 days.
For transplantation of tumor tissues instead of tumor cells, the frozen tissues were
thawed and washed in RPMI 1640 medium. About 2 to 3 pieces of Walker 256
tumor tissues were subcutaneously implanted into the right or left thigh of
anaesthetized female Sprague Dawley rats. The animals were observed for tumor
development and growth.
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3.2.2 MAINTENANCE OF THE CELL LINE
To maintain the cell line, a tumor weighing about 4 - 5 g was normally used. The
rats bearing the tumor were euthanized with an overdose of pentobarbital (250
mg/kg). The tumor was quickly excised; minced using blades and scissors and
filtered through a sterile piece of gauze. The cell suspension was centrifuged, the
supernatant decanted and the cell pellets re-suspended in an equal volume of freezing
medium containing RPMI 1640, fetal calf serum and dimethyl sulphoxide in the ratio
of 7:2:1 respectively. The cell suspension in the freezing medium was thoroughly
mixed by gentle agitation and gradually cooled. The cell suspension was first placed
at 4°C for two hours, at -20°C for 4 hours, then stored at -80°C in the deep freezer.
In addition to the storage of cell pellets, in some cases tumor tissues were also stored.
The non-necrotic areas of a 4-5 g tumor were cut into 3-6 mm fragments. The
fragments were washed in RPMI 1640 medium, and about 6-7 pieces of the tumor
tissues added to sterile vials containing the freezing medium (RPMI 1640 medium,
fetal calf serum, and dimethyl sulphoxide at 7:2:1 ratio). The vials were gradually
cooled, first at -20°C for 24 hours and the transferred to the deep freezer at -80°C for
long term storage.
3.3 WALKER 256 CARCINOMA CELLS FOR CELL CULTURE STUDIES
Walker 256 carcinoma cells for cell culture studies were purchased from the
American Type Culture Collection (ATCC) at cell passage number 290 and a density
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of 6.9* 106 cells/ml. Cells were cultured immediately upon receipt. They were grown
and maintained as monolayers in Medium 199 supplemented with 5% horse serum in
T-75 cm2 sterile flasks incubated at 37°C in 5% CO2. The culture medium (95%
Medium 199 and 5% horse serum) was replaced every 3-4 days and cells were sub
cultured when about 90% confluent (about 5 - 7 days).
3.3.1 SUB-CULTURE OF CELLS
Cells were sub-cultured when about 90-95% confluent. Basically, the culture
medium was decanted, the cell layer washed once with phosphate buffered saline
(PBS) pre-warmed to 37°C. Trypsin-EDTA (0.05% Trypsin, 0.53 mM EDTA«4Na)
solution warmed to 37°C was added to the cells to aid in their detachment from the
flask and removed immediately. The cells were then kept at room temperature for
about 5 minutes or until completely detached. Upon complete detachment of the cells
from the culture flask, culture medium pre-warmed to 37°C was added to the cells.
The cells in the medium were aspirated to dissociate cell aggregates, counted using
the Coulter Counter and dispensed into new flasks containing culture medium. Cells
were usually seeded at a density of about 3.5 * 106 cells/ml in sterile T-75cm2 flasks
for cell line maintenance and in T-225 cm2 flasks for transport studies. The flasks
were placed in an incubator at 37°C and 5% CO2 . All procedures were performed
under strict aseptic conditions. Cell viability was routinely tested using the trypan
blue exclusion test.
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3.3.2 MAINTENANCE OF THE CELL LINE
To maintain the cell line, when the cells were nearly confluent, the culture medium
was decanted. The cell layer was washed once with pre-warmed PBS. A trypsin-
EDTA (0.05% Trypsin, 0.53 mM EDTA®4Na) solution was added to the cells,
removed and the cells left at room temperature until completely detached (about 5
mins). Upon complete detachment of the cells from the flask, culture medium was
added to the cells and the cells vigorously aspirated to completely dissociate cell
aggregates. The cells were then counted using the Coulter Counter. The dissociated
cells were poured into a sterile centrifuge tube, centrifuged at 800 - 1 0 0 0 rpm for 1 0
minutes and the medium (supernatant) completely poured out. The cells were re
suspended in freezing medium made up of 95% culture medium and 5% dimethyl
sulphoxide (DMSO) and thoroughly mixed by gentle agitation. The freezing tubes
were properly labeled with name, date and cell density and to them were added about
1.5 to 1.8 ml of cells (usually about 2 - 2.5 * 106 cells/ml). Tubes were immediately
frozen at -70°C for the first 24 hours in a controlled cooling rate freezing unit. After
24 hours at -70°C, the vials were transferred to liquid nitrogen for long term storage.
The amount of freezing medium required was determined by multiplying the cell
density (cells/ml) (counting result from the Coulter Counter) with volume of the
culture medium (ml) added to the detached cells after trypsin treatment. The result
was the number of cells. This was then divided by the desired density of cells in each
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freezing vial (that is, the number of cells per ml of medium). This result was the
volume of freezing medium required to suspend the cells.
3.4 EX-VIVO AND IN-VITRO STUDIES
3.4.1 TRANSPORT STUDIES USING ANIMAL MODELS
Female Sprague Dawley rats weighing about 200 g and bearing the Walker 256
tumor of about 1 - 1.5 cm in size were used for the transport studies. The animals
were anaesthetized with a 1 : 1 mixture of ketamine ( 1 0 0 mg/ml) and xylazine
(Rompum, 20 mg/ml). 5FU was administered as an intravenous bolus dose (150
mg/kg and 30 mg/kg). Two hours after drug administration, the rats were euthanized
with an overdose of pentobarbital (250 mg/kg). The tumor, liver and thigh muscle
tissues were quickly excised and frozen till extracted by perchloric acid extraction.
At least 4 rats were used for each dosage level.
3.4.1.1 PERCHLORIC ACID EXTRACTION OF ANIMAL TISSUES
Tumor and liver tissues were weighed and thawed in 1 volume ice-cold 0.9 N
perchloric acid (PCA) and homogenized. The homogenate was thoroughly mixed in
ice for 10 minutes and then centrifuged at 1000 rpm for 10 minutes. The resultant
supernatant was the acid soluble fraction (ASF). The precipitate was washed twice
with ice cold 0.2 N perchloric acid, and the washings were added to the acid soluble
fraction. The acid soluble fractions were filtered using a 0.22p Millipore filter paper
prior to 1 9 F NMR analysis.
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3.4.1.2 ANALYSIS OF THE ACID INSOLUBLE FRACTION
To the tissue residue was added two volumes of 2 N KOH, mixed and incubated for
1 hour at 37°C after which the digest was cooled in ice. The protein and DNA were
precipitated by adding perchloric acid until the digest was acidic (pH 1-1.5) and left
to stand in ice for 10 minutes. The precipitate was separated by centrifugation and
the residue was washed twice with 0.2 N perchloric acid. The supernatant and the
washings were combined, neutralized with KOH and centrifuged to yield the final
supernatant as the RNA fraction.
The remaining residues were digested for 15 minutes at 60°C in 1 N perchloric acid
and centrifuged to yield the DNA fraction as the supernatant and the remaining
residue as the protein fraction.
3.4.2 TRANSPORT STUDIES USING CELL CULTURES
All transport studies were done in triplicates on nearly confluent (about 90%)
monolayer cells. Confluence was determined by visual inspection. All experiments
were carried out at 37°C, 5% CO2 and under strict aseptic conditions. Most of the
studies on the transport of 5FU were carried out using 1 mM 5FU concentration.
This concentration was selected to allow adequate signal-to-noise ratio to enable a
good detection of fluorinated signals while still within the region of non-saturation of
the transporter (s) involved in the transport process.
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For transport studies involving incubation of cells with 5FU and/or other agents in
the absence of extracellular Na+ , a bicarbonate Ringer’s buffer was prepared without
any added extracellular Na+ and the isomolarity of the medium maintained with
choline bicarbonate. The absence of extracellular Na+ means that no Na+ was
intentionally added to the incubation medium. Na+ being ubiquitous in nature can be
unavoidably present such as in the distilled water used in the preparation of the
buffers. For studies requiring the incubation of cells with a glucose free buffer, a
bicarbonate Ringer’s buffer was prepared without the addition of glucose. The pH of
all the media was balanced to between 1 2 -1 A and osmolarity balanced between 280
- 320 mOsm. Regular bicarbonate Ringer’s buffer was prepared with both
extracellular glucose and Na+.
3.4.2.1 A TYPICAL TRANSPORT STUDY USING CELL CULTURES
The culture medium was decanted and the cell layer was washed once with pre-
warmed serum free medium 199. The cells were incubated in a sufficient volume
serum free medium 199 for 1 hr. After the pre-incubation in serum free media, the
cells were then incubated with 5FU alone or with other agents for the desired length
of time in serum free medium 199 or in bicarbonate Ringer’s buffer as the case may
be. When the experiment called for the absence of extracellular Na+ or extracellular
glucose, the appropriate bicarbonate Ringer’s buffer was used for the incubation of
cells. At the end of the incubation time, uptake was rapidly stopped by a quick
“temperature drop” assay.
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Briefly, the incubation medium was quickly removed; the cell layer was washed
once with a sufficient volume (45 ml) of ice cold PBS and the cell layer was quickly
frozen over liquid nitrogen. The frozen cell layer was scrapped off the culture flask
using a cell scrapper and the cell mixture transferred with a pipette into a sterile 15-
ml centrifuge tube for acid extraction.
3.4.2.2 PERCHLORIC ACID EXTRACTION OF CULTURED CELLS
Two volumes of ice cold 0.9 M HCIO4 were added to the cell mixture in the
centrifuge flask. The mixture was left to sit in ice for 10-mins, and cooled further at
0°C for additional 10-mins and then centrifuged at 3000 g for 5 min at 4°C to yield
the acid soluble fraction (ASF) as the supernatant and the residue as the acid
insoluble fraction.
3.4.2.3 ANALYSIS OF THE ACID INSOLUBLE FRACTION
The residue was digested with two volumes of 2 N KOH for 1 hr at 37°C after which
it was cooled in ice. The mixture was acidified with 0.9 M HCIO4 until pH was
between 1-1.5 and then left in ice for 10 min. The solution was then centrifuged at
3000 g for 5 min to yield the supernatant as the RNA fraction. The residue was
washed with 0.9 M HCIO4, the washings added to the supernatant and the pH
neutralized with 2 N KOH. The remaining residue was digested with 1 N HCIO4 at
60°C for 15 min and the mixture centrifuged at 3000 g for 5 min yielding the
supernatant as the DNA fraction and the residue as the protein fraction. The protein
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fraction was dissolved in 1 N NaOH, the pH neutralized and the protein content of
the cells estimated using the bovine serum albumin (BSA), described below.
3.4.2.4 ESTIMATION OF THE CELL PROTEIN CONTENT
Development of a Calibration Curve:
The PIERCE BCA Protein Assay Kit was used for the protein analysis. A fresh set of
protein standards of known concentrations ( 2 0 - 2 , 0 0 0 fig/ml) were prepared by
diluting the 2.0 mg/ml BSA stock standard in phosphate buffered saline (PBS). The
working reagent (WR) was prepared by mixing 50 parts of BCA Reagent A (1,000
ml of reagent A contains sodium carbonate, sodium bicarbonate, bicinchoninic acid
and sodium tartrate in 0.1 M sodium hydroxide) with 1 part of BCA Reagent B (25
ml of a solution containing 4% cupric sulfate). Twenty-five pi of each protein
standard was pipetted into micro-well plate wells. 25 pi of PBS was used for the
blank wells. To each of the wells, was added 200 pi of the WR and the plate well
properly mixed on a plate shaker for 30 seconds. The plate was then covered and
incubated at 37°C for 30 minutes. After the incubation, the plate was cooled to room
temperature and the absorbency of each well was measured at 562 nm.
After the absorbency readings, the average readings for the blanks were subtracted
from the average readings for each of the standards. A calibration curve was
prepared by plotting the average blank corrected absorbency (@ 562 nm) for each
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BSA standard against its concentration in [ig/ml. The calibration curve was used to
determine the protein concentration for each unknown sample.
Estimating the Protein Content of Samples:
The protein residue obtained after the separation of the RNA and DNA fractions,
were dissolved in 1 volume of IN NaOH. The pH was neutralized and the samples
diluted in PBS. 25 pi of each unknown sample was pipetted into micro-well plate
wells and 25 pi of PBS used for the blank wells. 200 pi of the working reagent was
then added to each of the samples and the plate properly mixed on a plate shaker.
The plate was incubated at 37°C for 30 mins, cooled to room temperature and the
absorbency of each well measured at 562 nm. At least 3 samples of an unknown
sample were measured. Absorbency measurements were usually completed within 2
minutes after incubation. The average blank readings were subtracted from the
average readings of each of the samples. With the known absorbency of the samples,
the calibration curve was used to estimate the protein concentration of each of the
samples.
3.5 ANALYTICAL METHOD: IN-VITRO 1 9 F NMR SPECTROSCOPY:
All 1 9 F NMR spectra of the acid soluble fractions (ASF) of tissue and cell extracts
were measured using a GEMINI 200 Fourier Transform (FT) NMR spectrometer
operating at 200 MHz (Varian Associates, Palo Alto, CA). The magnet was tuned to
fluorine-19 and properly shimmed manually, to optimize data acquisition.
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The magnetic field was shimmed with the ]H NMR resonance of water observed in
the continuous wave mode. The magnetic field was also routinely shimmed and
quality control checked with a reference standard of 10 mg/ml 5FU. Spectra were
run with a lock solvent (D2O) and no proton decoupling in 10-mm diameter NMR
tubes.
For the 1 9 F NMR acquisitions of the acid soluble fractions from animal tissues, the
instrumental settings were established as follows: Sweep Width (SW) = 14,992.5 Hz,
Pulse Width (PW) = 26, TO = -9000; number of free induction decays (FIDs);
84,000 and no delay time. Under these conditions, the peak signal due to 5FU was
measured at a chemical shift of about 34.6-34.8 ppm. The instrumental settings for
the 1 9 F NMR acquisitions of the acid soluble fractions from cell extracts, were
established as follows: Sweep Width = 19,011.4 Hz; Pulse Width = 26; TO = -9000,
number of free induction decays (FIDs) = 28,000 and a delay time (Dl) of 5s. At the
wider window of SW = 19,011.4 Hz, the chemical shift of 5FU was measured at
about 45.5 ppm.
Calibrated reference standards of 5FU were used to allow the quantitation of the
intracellular concentration of 5FU in the ASF of animal tissues and cell extracts. A
2-mm capillary tube was filled with 0.5 ml of a 1-mM solution of 5-Fluoro-DL-
tryptophan and used as an external reference standard. This external reference
standard was calibrated against known concentrations of 5FU solutions.
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The calculated intracellular concentration of 5FU was normalized by the cell protein
content, which was estimated using the bovine serum albumin (BSA).
3.5.1 QUANTITATION FOR IN- VITRO 1 9 F NMR SPECTROSCOPY
To optimize for maximum sensitivity and quantitation, data acquisition parameters
were carefully selected.
3.5.1.1 SIGNAL SATURATION FACTOR
To determine the optimum pulse repetition time, an array experiment was used to
calibrate the delay time (Dl). Spectra of 5FU (1 mM) were acquired at pulse
repetition intervals of between 1 second and 15 seconds using the same parameters
and number of field induction decays (FIDs). The number of acquisitions used (NT)
was 1. The 5FU peak had its highest intensity at 5 seconds delay time. Therefore, a 5
seconds pulse repetition time was used to allow a good relaxation of the 5FU
resonance.
3.5.1.2 PULSE WIDTH CALIBRATION
In other to determine the optimum pulse width (PW) for the detection of 5FU signal,
an array experiment was performed. 1 9 F NMR spectra of 1 mM 5FU was acquired at
different pulse widths, NT = 1 and Dl = 5 sec. The initial sets of PW values used
were 10, 20, 30, 40, 50, 60, 70 and 80. After acquisitions of 1 9 F NMR spectra of the
1-mM sample of 5FU at the different PW values, the optimum PW value, which was
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the value that gave the highest 5FU peak intensity, was between 20 and 30. Another
array experiment was performed with PW values 20, 21, 22, 23, 24, 25, 26, 27, 28,
29 and 30 and using the same acquisition parameters. The optimum PW value was
calculated to be 26, as it gave the highest intensity of the 5FU signal. Therefore, all
1 9 F NMR acquisitions were done using a PW of 26.
3.5.2 QUANTITATIVE CALIBRATION OF 5FU IN TISSUE EXTRACTS
To enable the quantitation of 5FU in the tissue extracts, a calibration curve for 5FU
was developed by using the ASF (to enable calibration of the paramagnetic materials
present in the tissue extracts) of tissue extracts from control rats. Control rats were
tumor-bearing rats that received no administered dose of 5FU. Different known
concentrations of 5FU (0.05, 0.1, 0.5, 1.0, 2.5, 5.0, and 10.0 mg/ml) were added to
the ASF of tumor and liver tissues from these control rats.
1 9 F NMR spectra of each of the solutions were acquired under the following
conditions: Sweep Width = 14,992.5 Hz, Pulse Width = 26, TO = -9000, NT = 1,000
and Dl = 0. The peak heights, peak areas and signal-to-noise (S/N) ratio (of a
specific region on the spectra) of the various known concentrations of 5FU were
electronically calculated. Plots of the peak heights, peak areas and S/N ratio against
the concentrations of 5FU were all linear with R2 of 0.998, 0.992 and 0.996
respectively.
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The relative amounts of 5FU in the tissue extracts were estimated using the
following formula;
Relative amounts of 5FU = (Peak Height * S/N)/2.5
A calibration curve was developed by plotting the relative amounts of 5FU expressed
in S/N, calculated form the above formula against the known concentrations of 5FU.
The calibration curve showed a linear relationship between the S/N ratio estimate of
the amount of 5FU in the tissue extracts and the concentrations of 5FU (R2 = 0.992).
A linear equation was obtained according to the formula:
Y = mx + b
The developed calibration curve and the linear equation were used to estimate the
amount of 5FU in the acid soluble fractions of the tumor and liver tissue extracts
from rats that had received 5FU. The estimated amounts of 5FU in the tissue extracts
were normalized by the weight of the tissues.
3.5.3 QUANTITATIVE CALIBRATION OF CELL EXTRACTS
To allow for quantitation of intracellular 5FU in the acid soluble fraction of cell
extracts, a calibration curve of 5FU was developed against known concentrations of
5FU. The external reference standard (0.5 ml of 1 mM 5-Fluoro-DL-tryptophan in a
2-mm capillary tube) was calibrated against known concentrations of 5FU.
53
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The calibration curve was developed by preparing different concentrations of 5FU
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5 and 2.0 mM concentrations in both
Medium 199 and bicarbonate Ringer’s buffer containing both extracellular Na+ and
glucose. 1 9 F NMR spectra were obtained of each of the standard solutions of 5FU
under the following instrumental settings: Sweep Width (SW); 19,011.4 Hz, Pulse
Width (PW); 26; Dl = 5 s; FIDs = 1,000. Based on the wider window of SW:
19,011.4 Hz, the 5FU signal was measured at a chemical shift of about 45.3 ppm. All
data acquisitions were carried out at room temperature.
The peak intensities, peak areas and signal-to-noise ratio (a specified region on the
spectra) corresponding to the various concentrations of 5FU and those of 5-fluoro-
tryptophan were electronically calculated.
A plot of the ratio of the peak areas of 5FU and the reference standard, as a function
of the concentration of 5FU in mM was linear. In addition, plots of the signal-to-
noise ratios and the peak intensities of 5FU against the concentration of 5FU in mM
were also linear, R2 = 0.986.
The following formula was used to calculate the relative amounts of 5FU.
Relative amounts of 5FU = (Peak Height * S/N)/2.5
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A Calibration curve was developed by plotting the calculated relative amount of 5FU
against the concentration of the standard solutions (mM). This plot gave a linear
relationship with an R2 of 0.992.
The relative amounts of 5FU in the acid soluble fraction (ASF) of the cell extracts
were estimated using the above formula and the concentrations estimated using the
developed calibration curve and the obtained linear equation. The amounts of 5FU in
the cell extracts were expressed as pg, by multiplying the concentration in mM by
the volume and molecular weight of 5FU. The calculated amounts of 5FU in the ASF
of the cell extracts were normalized by the amount of protein in the cell and
expressed as pg per mg of protein.
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CHAPTER IV
4.0 RESULTS
STUDIES ON THE TRANSPORT OF 5FU INTO
WALKER 256 TUMOR CELLS
The following are results from studies undertaken in animal models bearing the
Walker 256 adenocarcinoma and cell cultures of the same tumor type to understand
the transport and regulation of transport of 5FU into tumor cells.
4.1 ANIMAL STUDIES
4.1.1 ACCUMULATION OF FREE 5FU IN THE TUMOR ASF
The dose dependent uptake of 5FU into tumor cells after an intravenous
administration of a bolus dose of 5FU (150 mg/kg and 30 mg/kg) to rats bearing the
Walker 256 tumor was studied. At 2 hours post drug administration, free 5FU was
present as the major component in the tumor acid soluble fraction at the two different
doses studied (Figure 4.1).
Figure 4.1 is a representative spectrum of the acid soluble fraction (ASF) of tumor
tissue extracts from a rat that had been administered an intravenous bolus dose, 150
mg/kg of 5FU and euthanized 2 hours post drug administration. The predominant
component of the ASF, free 5FU was measured at a chemical shift of about 34.8
56
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ppm. The amount of free 5FU measured in the ASF of rat tumors, following
administration of two widely different doses (30 mg/kg and 150 mg/kg) of 5FU, is
consistent with a non-linear relationship. Between the administered dose of 5FU and
intracellular amounts measured in the tumor ASF, a 5 fold increase in the
administered dose of 5FU did not result in a concomitant increase in the amount of
intracellular free 5FU measured in the tumor ASF. Rather, it translated only into a
50% increase in intracellular free 5FU measured in the tumor ASF (Table 4.1).
There were significant amounts of nucleotides and nucleosides of 5FU in the tumor
tissue extracts and equally significant amounts of a-fluoro-B-alanine (FBAL), the
major catabolite of 5FU in the tumor ASF, 2 hours post drug administration (Fig.
4.1). As seen in Figure 4.1 significant amounts of fluoronucleotides of 5FU were
measured at a chemical shift of about 42.3-ppm, 7 - 8 ppm downfield from 5FU. The
fluoronucleosides of 5FU were measured at a chemical shift range of about 38.2-38.6
ppm, ranging about 3 - 4 ppm downfield from 5FU. Due to the poor resolution of the
magnet, it was not possible to distinguish between the various fluoronucleotides and
fluoronucleosides present.
FBAL was measured at a chemical shift of about 8.5-ppm, about -26-ppm upfield
from 5FU. At neutral pH (pH = 7.0), FBAL was detected at about -19 ppm upfield
from 5FU. It can be noted that the phasing of the 5FU and the fluoronucleotide and
fluoronucleoside signals are different from that of FBAL, due to the adjacent protons
5 7
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on the 5FU molecule. FBAL is phased in the opposite direction from the signals of
5FU, the fluoronucleotides and the fluoronucleosides. Due to the acid environment
of the ASF (pH = 1.2 - 1.8), the chemical shift of FBAL shifted by about 7 ppm
further up causing the signal due to FBAL to appear at a chemical shift of about -26
ppm upfield from 5FU.
Dose (mg/kg) 5FU Nucleosides Nucleotides FBAL
150 7.7 ±0.39 5.0 ±0.2 4.5 ±0.14 5.1 ±0.3
30 5.3 ±0.21 2.9 ±0.14 2.9 ±0.10 2.5 ±0.11
Table 4.1: Free 5FU and its metabolites in the tumor ASF of rats, expressed as
relative units (S/N).
Rats were administered two different doses of 5FU (30 mg/kg and 150 mg/kg) as
intravenous bolus doses, euthanized 2 hours post drug administration and tumor
tissues quickly excised. Intracellular amounts of the fluorinated compounds in the
tumor were measured as previously described in “Materials and Methods”. Results
are the average of measurements from 4 rats at each dose of 5FU. Amounts of 5FU
and metabolites are expressed per g of tissue.
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Figure 4.1: A representative 1 9 F spectrum of the tumor ASF from rats 2 hours after
administration of 150 mg/kg 5FU. The 1 9 F NMR settings were as follows: SW =
14,992.5 Hz, TO = -9000, PW = 26, NT = 84,000, D1 = 0. The peaks were identified
using 5FU as the reference peak.
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4.1.2 ACCUMULATION OF FREE 5FU IN THE LIVER ASF
The dose dependent uptake of 5FU into the liver tissues of rats after administration
of two different doses (150 mg/kg and 30 mg/kg) of 5FU was studied. 2 hours after
drug administration, free 5FU was barely detected in the liver ASF. On the other
hand, FBAL was measured as the major component of the liver ASF (Figure 4.2).
Figure 4.2, shows a typical spectrum of the liver ASF after 2 hours of drug
administration from rats that had received 150 mg/kg 5FU. Using 5FU as a reference
peak, FBAL was measured at about 8.5 ppm, about -26 ppm upfield from 5FU (pH
= 1.2 - 1.8). There were detectable amounts of fluoronucleotides, anabolites of 5FU,
measured at about 42.5 ppm, 7 - 8 ppm, downfield from 5FU but no
fluoronucleosides were detected in the liver ASF (Figure 4.2). .
At the 2 different doses (150 mg/kg and 30 mg/kg) of 5FU studied, the amounts of
FBAL measured in the liver was about 5 times the amount measured in the tumor
ASF (Table 4.1 & 4.2). A five-fold increase in the administered dose of 5FU,
doubled the amount of FBAL detected in the liver (Table 4.2).
Figure 4.3, clearly identifies the peak at 8 .5-ppm chemical shift as the signal due to
FBAL.
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Dose (mg/kg) 5FU Nucleosides Nucleotides FBAL
150 <1.5 1.7 ±0.08 4.2 ±0.12 25 ± 1.5
30 <1.5 < 1.5 2.2 ± 0.07 11.25 ±0.5
Table 4.2: 5FU and its metabolites in the liver ASF of rats, expressed as relative
units (S/N).
Rats were administered two different doses of 5FU (30 mg/kg and 150 mg/kg) as
intravenous bolus doses, euthanized 2 hours post drug administration and liver
tissues quickly excised. Intracellular amounts of the fluorinated compounds were
measured as previously described in “Materials and Methods”. Results are the
average of measurements from 4 rats. Amounts of 5FU and metabolites are
expressed per g of tissue.
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Figure 4.2: A representative 1 9 F spectrum of the liver ASF from rats 2 hours after
administration of 150 mg/kg 5FU. The 1 9 F NMR data were obtained using the
following parameters: SW - 14,992.5 Hz, TO = -9000, PW = 26, NT - 84,000, D1 -
0. The various peaks were identified using 5FU as the reference peak.
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Figure 4.3: A representative 1 9 F spectrum of FBAL in the liver ASF.
The presence of the six-split peaks clearly identifies FBAL measured at a chemical
shift of about 8 ppm, about -26 ppm upfield from 5FU, the major component of the
liver ASF. The peak due to FBAL was identified using 5FU as the reference peak.
63
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4.2 CELL CULTURE STUDIES
4.2.1 ACCUMULATION OF FREE 5FU IN THE WALKER 256 TUMOR
CELLS
The accumulation of free 5FU in the Walker 256 cells as a function of time was
studied in the acid soluble fraction (ASF), as there were no detectable 1 9 F signals in
the RNA and DNA fractions even after 90-min of incubation. The 1 9 F NMR analysis
of the ASF at the indicated time points in the presence of extracellular glucose and
Na+ indicate that most of the 5FU transported intracellularly was present as free 5FU
(Figure 4.4). Figure 4.4 & 4.5 are representative spectra of 5FU measured in the acid
soluble fraction of cells exposed to 1 mM 5FU in the presence of extracellular
glucose and Na+ for 1 and 1 0 -min respectively.
At 1-minute incubation time, free 5FU was the only 1 9 F signal detectable in the ASF
of tumor cell extracts, appearing at a chemical shift of about 34.7 ppm (Figure 4.4),
(5FU reference standard measured a chemical shift range 34.6 - 34.8 ppm when SW
is set at 14,992.5 Hz). At 10 minutes of incubation of cells in the presence of glucose
and extracellular Na+ , there were still no detectable metabolites of 5FU in the ASF of
the cell extracts (Figure 4.5). There were no detectable metabolites of 5FU in the
ASF until after about 90 minutes of incubation of Walker 256 cells with 1 mM 5FU
in the presence of extracellular glucose and Na+ (Figure 4.6). Figure 4.6 shows
detectable amounts of the fluoronucleosides (anabolites of 5FU) of 5FU measured at
a chemical shift of about 49 ppm, about 3 - 4 ppm downfield from 5FU.
64
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Figure 4.7 shows the accumulation of free 5FU into the Walker 256 cells as a
function of time. The intracellular transport of 5FU was time dependent and showed
a rapid initial uptake, within the first 1-min of incubation. The accumulation of 5FU
into these cells continued to increase for up to 1 0 -min incubation time, beyond which
continued incubation failed to show an increase in the intracellular amount of free
5FU measured. Rather, the free 5EU signal showed a decrease in the intracellular
compartment with increased incubation.
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jjiM itt* iXi I Ji*ii j ikUiiA v J.l.iJ ill. iiUiit
70 8 0 50
T np»invTifiw'ifi|ii r*<«iriw r r f i r i ' 1 v t t
30 20 10 m 0
Figure 4.4: A representative 1 9 F spectrum of the ASF of cells after exposure to 1 mM
5FU for 1-min.
Walker 256 cells were exposed to 1 mM 5FU in bicarbonate Ringer’s buffer
containing both extracellular glucose and Na+ for 1-min. 1 9 F NMR Spectrum was
obtained using the following parameters: SW = 14,992.5 Hz, TO = - 9000, PW = 26,
NT = 28,000, D1 = 5 sec.
66
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Figure 4.5: A representative 1 9 F spectrum of the ASF of cells after exposure to 1 mM
5FU for 10-min.
Walker 256 cells were exposed to 1 mM 5FU in bicarbonate Ringer’s buffer
containing both extracellular glucose and Na+ for 10-min. Spectrum was obtained
using the following parameters: SW = 14,992.5 Hz, TO = - 9000, PW = 26, NT =
28,000, D1 = 5-sec.
6 7
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Figure 4.6: A representative 1 9 F spectrum of the ASF of cells after exposure to 1 mM
5FU for 90-min. Spectrum was obtained using the following parameters: SW =
19,011.4, TO = - 9000, PW - 26, NT = 28,000, D1 = 5 sec. At a wider window of
SW = 19,011.4, the 5FU signal was measured at a chemical shift range of 45.3 - 45.4
ppm.
68
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80 100
Figure 4.7: Accumulation of free 5FU into the Walker 256 tumor cells as a function
of time.
Walker 256 tumor cells were incubated with 1 mM 5FU in bicarbonate Ringer’s
buffer containing extracellular glucose and Na+ at 37°C for the indicated lengths of
time. At each indicated time point, the uptake was stopped and intracellular
accumulation of free 5FU in the ASF of cell extracts was measured as described in
“Materials and Methods”.
69
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4.3 RATIO OF INTRACELLULAR AND EXTRACELLULAR 5FU:
Figure 4.8 shows the distribution ratio of 5FU transported intracellularly against the
concentration of 5FU in the extracellular medium. 5FU accumulated intracellularly
against its concentration gradient. In less than 1-min of incubation of cells with 5FU,
the concentration of 5FU measured intracellularly equilibrated with the extracellular
concentration. After 1-min of incubation of cells with 1 mM 5FU in the presence of
extracellular glucose and Na+, the amount of 5FU measured intracellularly exceeded
the concentration of 5FU in the incubation medium, showing a 2:1 ratio. With
increased incubation time, 5FU continued to accumulate intracellularly against its
concentration gradient.
By 10-min of incubation with 1 mM 5FU, the amount of free 5FU measured
intracellularly was almost 3.5 times the concentration of 5FU in the incubation
medium. Beyond 10-min of incubation, 5FU still accumulated intracellularly against
its concentration gradient though the intracellular amounts measured never exceeded
the intracellular concentration at 10-min. For up to 90-min of incubation, the amount
of 5FU measured intracellularly remained above the concentration in the
extracellular medium.
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Time (mm)
Figure 4.8: Ratio of intracellular/extracellular 5FU (5FUin t/e x t)
Method is the same as described in legend for figure 4.7. The intracellular
concentration of 5FU was obtained by dividing the intracellular amount of 5FU by
the volume of the cell pellets. The ratio of 5FU concentration in the intracellular
compartment against that in the extracellular compartment (5FUjn t/e x t) was obtained
by dividing the intracellular concentration of 5FU at each time point with the
concentration of 5FU in the incubation medium.
71
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4.4 EFFECT OF EXTRACELLULAR Na+ ON 5FU TRANSPORT
4.4.1 5FU Transport in the absence o f extracellular Na+ - The effect of Na+ on the
transport of 5FU was investigated by studying the kinetics of the intracellular
accumulation of free 5FU (1 mM) into the Walker 256 cells in the absence of
extracellular Na+ at various time points (1, 5, 10, 20 and 30-min). When comparing
the kinetic studies of 5FU transport in the presence and absence of extracellular Na+,
no significant difference was measured at accumulation times 5,10,20 and 30-min
(Fig. 4.9).
The intracellular accumulation of 5FU in the absence of extracellular Na+ showed the
same pattern as uptake in the presence of Na+, showing a linear intracellular transport
for up to 10-mins. Continued incubation failed to increase the amount of free 5FU
measured intracellularly (Fig. 4.9) but rather showed a decrease in the free 5FU
measured intracellularly.
The initial rate of uptake (@ 1 min incubation time) of 5FU into the cells was
slightly decreased by about 30% in the absence of extracellular Na+ when compared
to the intracellular accumulation of 5FU in the presence of Na+ at the same
accumulation time (Fig. 4.9). Figure 4.10 is a representative spectrum of 5FU
measured in the ASF of the Walker 256 cells after a 1-min exposure in a Na+ free
buffer. Intracellular free 5FU was measured at a chemical shift of about 34.7 ppm
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and shows the slight inhibition of 5FU uptake into the cells at 1 min incubation time
in the absence of extracellular Na+.
Therefore, the presence of extracellular Na+ in the incubation medium slightly
stimulated the initial uptake of 5FU in the first minute of incubation with the
disappearance of the inhibitory effect as cells were incubated for longer periods of
time with 5FU (Fig. 4.9). By 5-min incubation time, the intracellular accumulation of
5FU closely resembled the accumulation of 5FU in a Na+ containing medium (Figure
4.9). Figure 4.11 is a representative spectrum of 5FU measured in the ASF of cells
incubated with 1 mM 5FU in a Na+ free buffer for 5-min, with free 5FU measured at
a chemical shift of about 34.7 ppm.
5FU accumulated intracellularly against its concentration gradient even in the
absence of extracellular Na+ (Figure 4.12). At 5-min, the intracellular concentration
5FU measured exceeded the concentration of 5FU in the incubation medium with a
ratio of 3.3:1 even in the absence of extracellular Na+. Likewise, the intracellular
concentration of 5FU remained above the extracellular concentration for 30-min of
incubation, with the highest intracellular/extracellular ratio (4:1) measured at 10-min.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Figure 4.9 - Time course of 5FU Transport into Walker 256 tumor cells in the
presence and absence of extracellular Na+: Accumulation of free 5FU into the ASF
of Walker 256 cells in the presence and in the absence of extracellular Na+ at the
indicated time points was measured in the ASF. Walker 256 cells were incubated
with 1 mM 5FU for the indicated times at 37°C in bicarbonate Ringer’s buffer
containing Na+ or with no added extracellular Na+. The intracellular accumulation of
5FU was measured as previously described in “MATERIALS AND METHODS”.
(♦) Represents uptake in the ASF in the presence of Na+ and (□) represents uptake in
the absence of extracellular Na+.
7 4
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Figure 4.10: A representative 1 9 F spectrum of the ASF of cells after exposure to 1
mM 5FU in Na+ free buffer for 1-min. Spectrum was obtained using the following
parameters: SW - 14,992.5 Hz, PW = 26, TO = - 9000, NT = 28,000, D1 = 5 sec.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4.11: A representative 1 9 F spectrum of the ASF of cells after exposure to 1
mM 5FU in Na+ free buffer for 5-min. Spectrum was obtained using the following
parameters: SW = 14,992.5 Hz, PW = 26, TO - - 9000, NT = 28,000, D1 = 5 sec.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Figure 4.12 - Ratio of 5FU(intracellular/extracellular concentration) in the absence of
extracellular Na+.
Walker 256 cells were incubated with 1 mM 5FU in Na+ free bicarbonate Ringer’s
buffer at the indicated times. The intracellular concentration of 5FU at each time
point was calculated by dividing the intracellular amount of 5FU by the volume of
the cell pellets. The ratio of 5FU concentration in the intracellular compartment
against that in the extracellular compartment (5FUmt/ext) was obtained by dividing the
intracellular concentration of 5FU at each time point with the concentration of 5FU
in the incubation medium.
77
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4.4.2 Effect o f ouabain treatment on the transport o f 5FU:
The initial intracellular transport of 5FU into the Walker 256 tumor cells at 1-min
incubation time was slightly inhibited by about 25% when the cells were pre
incubated with 1 mM ouabain for 30-mins. With increased incubation of cells for up
to 10 min with 1 mM 5FU after pre-treatment with ouabain, the inhibitory effect
disappeared and there were no measured difference in the intracellular transport of
5FU between uptake in the presence or absence of ouabain pre-treatment (Table 4.3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Length of
incubation 5FU 5FU +
Ouabain
% Inhibition1
Intracellular 5FU
(pg/mg protein) 1 -min 88.4 ±6.50 66.2 ± 4.20 25.06
Intracellular 5FU
(pg/mg protein) 10-mins 144.8 ±7.50 154.4 ± 15.0 -
Table 4.3: Effect of ouabain treatment on the transport of 5FU into Walker 256
tumor cells. Walker 256 cells were pre-incubated with 1-mM ouabain for 30 min at
37°C in glucose and Na+ containing bicarbonate Ringer’s buffer. 1 mM 5FU was
then added to the incubation medium and incubation continued under the same
conditions for additional times (1 and 10-min). Intracellular uptake of 5FU into the
ASF of the cell extracts was measured as previously described. x % Inhibition is
expressed in comparison with uptake of 5FU in the absence of ouabain at the same
accumulation time.
7 9
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4.5 EFFECT OF GLUCOSE ON 5FU TRANSPORT
4.5.1 5FU transport in the absence o f extracellular glucose:
The intracellular transport of 5FU (1 mM) into the Walker 256 cells was measured in
glucose free bicarbonate Ringer’s buffer at 10-min accumulation time. The transport
of 5FU under this condition, showed a 30.4 % inhibition in 5FU transport (Table
4.4). Pre-incubating the cells in glucose free bicarbonate Ringer’s buffer for 30-min
prior to the addition o f 1 mM 5FU into the incubation medium, showed a slightly
higher inhibition of 5FU transport (36.53%) at 10-min accumulation time.
There was no significant difference in the inhibition of intracellular accumulation of
5FU when the cells were pre-incubated in glucose free buffer for 30-min and when
the transport of 5FU was measured directly in the glucose free buffer without prior
incubation. Pre-incubating the cells in glucose free buffer prior to the addition of
5FU resulted only in a 6% increase in the inhibition of 5FU transport when
compared to the intracellular transport of 5FU into the cells without prior pre
incubation in glucose free buffer.
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Intracellular 5FU
(pg/mg protein)
% Inhibition1
Regular buffer 144.8 ±7.50
Glucose free buffer
(No pre-incubation)
100.8 ±8.10 30.39
Glucose free buffer
(Pre-incubated)
91.9 ±9.20 36.53
Table 4.4: Effect of Extracellular Glucose on 5FU Transport
Walker 256 cells were incubated with 1 mM 5FU in glucose free buffer for 10-min
with or without pre-incubation in glucose free bicarbonate Ringer’s buffer for 30-
min. Intracellular accumulation of free 5FU was measured as previously described.
l% Inhibition is expressed in comparison with transport in the presence of
extracellular glucose.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Figure 4.13: Effect of Extracellular Glucose on 5FU Transport. Walker 256 cells
were incubated with 1 mM 5FU for 10 min at 37°C in glucose-free bicarbonate
Ringer’s buffer. Intracellular 5FU was measured in the ASF as previously described.
% Inhibition is expressed in comparison with intracellular accumulation of 5FU in
the presence of extracellular glucose at the same accumulation time. (Cells were not
pre-incubated in glucose free buffer).
8 2
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Figure 4.14: Effect of Pre-incubation of Cells in Glucose-free buffer on the Transport
of 5FU. Cells were pre-incubated in glucose free bicarbonate Ringer’s buffer at 37°C
for 30 minutes prior to the addition of 1 mM 5FU. The cells were then exposed to 1
mM 5FU in the glucose free bicarbonate Ringer’s buffer for 10 minutes. The
intracellular uptake of 5FU was measured as previously described. % Inhibition is
expressed in comparison with 5FU uptake in the presence of extracellular glucose.
83
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4.5.2 Effect o f a glycolytic inhibitor on the transport o f 5FU:
The transport of 1 mM 5FU into Walker 256 cells at 10-mins accumulation time
after pre-incubation of cells for 30-min with 1 mM iodoacetate in glucose containing
bicarbonate Ringer’s buffer showed a 44% inhibition when compared to transport in
the absence of the glycolytic inhibitor.
5FU alone 5FU +
Iodoacetate
% Inhibition
Intracellular 5FU
(pg/mg protein)
144.8 ±7.5 81.4 ±9.2 43.80
Table 4.5: Effect of a Glycolytic Inhibitor, Iodoacetate, on 5FU Transport. Walker
256 cells were pre-incubated in 1 mM iodoacetate in glucose containing bicarbonate
Ringer’s buffer for 30-min. 1 mM 5FU was added to the incubation medium and
incubated with cells for 10-min. Accumulation of 5FU in the cells was measured as
stated previously. % Inhibition is expressed in comparison with the transport of 5FU
in glucose containing buffer in the absence of iodoacetate at the same accumulation
time.
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4.6 Effect o f extracellular pH on the transport o f SFU:
The transport of SFU into the Walker 256 tumor cells was measured at a lower
extracellular pH environment (pH = 6.7) in glucose containing bicarbonate Ringer’s
buffer. At extracellular pH 6.7, the intracellular transport of 1 mM SFU was
enhanced by 38% (Table 4.6 & Fig. 4.15) when compared to its intracellular
transport at pH 7.2, at the same accumulation time.
Intracellular 5FU
(pg/mg protein)
Extracellular pH = 7.2 144.8 ± 7.5
Extracellular pH = 6.7 235.5 ± 17.0
% Increase1 38.51
Table 4.6: Effect of Extracellular pH on SFU Transport.
Method is the same as the legend for figure 4.15.
1 % Increase is expressed in comparison with intracellular accumulation of 5FU in a
similar buffer at pH 7.2.
85
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Buffer
Figure 4.15: Effect of extracellular pH on 5FU transport.
Walker 256 cells were pre-incubated for 30 min in extracellular pH 6.7 in glucose
containing bicarbonate Ringer’s buffer prior to the addition of 1 mM 5FU to the
incubation medium and incubated with cells for 10-min. The intracellular
accumulation of SFU in a similar medium at 10-min incubation time was measured
at pH 7.2 and compared to the accumulation at pH 6.7.
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Figure 4.16: A Representative 1 9 F spectrum of the ASF of cells after at 10-min with
5FU in extracellular pH 6.7. Spectrum was obtained using the following parameters:
SW = 14,992.5; PW = 26; TO = -9000, NT = 28,000, D1 = 5 sec.
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4.7 EFFECT OF METABOLIC INHIBITORS ON 5FU TRANSPORT
4.7.1 Effect o f 2,4-dinitrophenol on SFU transport:
The effect of the uncoupling agent 2,4-dinitrophenol (DNP) (1 mM), on the transport
of 1 mM SFU into the Walker 256 cells was studied by measuring the transport of
5FU in the cells after pre-incubating cells with 1 mM 2,4-dinitrophenol in glucose
and Na+ containing medium for 30 minutes. 2,4-dinitrophenol failed to inhibit the
transport of 5FU into the Walker 256 cells.
SFU alone SFU + DNP
Intracellular SFU 144.8 + 7.5 154.2 ±4.2
(p g /m g protein)
Table 4.7: Effect of 2,4-dinitrophenol on the transport of SFU.
Walker 256 cells were pre-incubated with 1 mM DNP in glucose and Na+ containing
medium for 30 minutes at 37°C. After the pre-incubation, 1 mM SFU was added to
the incubation medium and cells incubated with SFU for additional 10 minutes. The
intracellular transport of SFU at 10-mins was measured as previously described.
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4.8 Kinetics o f SFU Uptake
The initial rate of 5FU influx into the cells, as a function of its concentration in the
incubation medium was determined and a hyperbolic saturation curve was obtained.
The Km value for 5FU graphically determined from the plot of rate of influx against
its concentration was 0.55 mM and the Vmax value was obtained to be 150.0 pg/mg
protein/min.
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0.00 J
0.0 0.5 2.0 2.5
[5FU], mM
Figure 4.17: 5FU uptake as a function of its concentration.
Walker 256 cells were incubated with different concentrations of 5FU (0.1, 0.5, 1.0,
1.5 and 2.0 mM) in bicarbonate Ringer’s buffer containing extracellular glucose and
Na+ for 1-min. The intracellular accumulation of 5FU into the Walker 256 cells at 1-
min was measured in the ASF.
89
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4.9 EFFECT OF STRUCTURALLY REALATED ANALOGS ON THE
TRANSPORT OF SFU INTO WALKER 256 TUMOR CELLS.
The transport of 1 mM SFU into the Walker 256 cells was measured in the presence
of indicated concentrations of structurally related compounds, uracil, thymine,
cytosine and uridine. After incubating cells for 10-mins with 1 mM 5FU and each of
the nucleobase analogs uracil, thymine and cytosine, uracil inhibited 5FU transport
into these cells by 11.5 % while thymine more substantially inhibited the transport of
5FU by 34.4 % . On the other hand, cytosine failed to inhibit the transport of SFU
into the Walker 256 cells (Table 4.8).
The nucleoside analog, uridine inhibited 5FU transport by almost 50% within 10
minutes of incubation. It did so more strongly than the pyrimidine bases (uracil and
thymine) inhibited the transport of 5FU. Cytidine equally inhibited 5FU transport by
53 % (Table 4.8).
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Addition Cone. (mM) Intracellular SFU
(pg/mg protein)
%
Inhibition1
None - 144.8 ±7.5 -
Cytosine 10 143.2 ±0.2 1.09
Uracil 10 128.2 ±6.5 11.46
Thymine 10 94.9 ±5.3 34.42
Uridine 10 74.0 ±4.5 48.88
Cytidine 10 67.6 ± 11.20 53.42
Table 4.8: Effect of structural analogs on the transport of 5FU.
Walker 256 cells were incubated with 1 mM 5FU and each of the analogs at the
indicated concentrations simultaneously in glucose and Na+ containing Medium 199
at 37°C for 10 min. Intracellular accumulation of 5FU into the ASF of the Walker
256 cells was measured. % Inhibition is expressed in comparison with uptake of 5FU
in the absence of an analog at the same accumulation time.
91
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STUDIES ON THE NATURE AND SPECIFICITY OF THE SFU
TRANSPORTER (S)
The following are results from studies on the nature and specificity of the transporter
(s) that mediate (s) 5FU transport into Walker 256 tumor cells.
4.10 COMPETITIVE INHIBITION OF SFU TRANSPORT BY URIDINE
The initial rate of influx of different concentrations (0.5, 1.0 and 1.5 mM) of 5FU
into the Walker 256 cells was measured in the presence of the different
concentrations of uridine (5,10, and 20 mM). The intracellular transport of 5FU was
increasingly inhibited as the concentration of uridine was increased. The transport of
0.5 mM 5FU into the cells was inhibited by 30 % in the presence of 5-mM
concentration of uridine. While at 10 mM uridine concentration, the transport of the
same concentration of 5FU (0.5 mM) was more greatly inhibited by more than 45%
(Table 4.9). At 20 mM concentration of uridine, there was an almost 60% inhibition
of the initial intracellular transport of 0.5 mM 5FU. 5 mM uridine inhibited the initial
transport of 1 mM SFU by 22% with the inhibition increasing to as high as 54%
within the first minute of incubation in the presence of 20 mM uridine (Table 4.9).
On the other hand, uridine became less inhibitory of 5FU transport as the
concentration of SFU in the incubation medium was increased. At 0.5 mM 5FU, 5
mM uridine inhibited the initial transport of 5FU by almost 30%. Whereas at 1.5 mM
9 2
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concentration of 5FU, 5 mM uridine inhibited the intracellular transport of SFU by
20% (Table 4.9). 10 mM uridine inhibited the intracellular transport of 0.5 mM 5FU
by 45% while the same concentration of uridine (10 mM) inhibited the intracellular
transport of 1.5 mM 5FU by 34%.
Figure 4.18, shows the steady decline in the amount of 5FU transported
intracellularly as the concentration of uridine in the incubation medium was
increased.
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Rate of SFU Uptake (pg/mg protein/min)
Uridine
(mM)
SFU
(0.5 mM)
%
Inhibition
SFU
(1 mM)
%
Inhibition
SFU
(1.5 mM)
%
Inhibition
0 62.0 ±6.0 94.7 ±6.2 122.9 ±8.2
5 43.6 ±4.2 29.76 73.2 ±5.1 22.72 97.6 ±6.1 20.59
10 33.7 ± 3.1 45.65 60.1+4.2 36.52 81.0 ±5.9 34.09
20 25.0 ±2.0 59.68 43.1 ±2.7 54.46 60.0 ± 4.2 51.18
Table 4.9: Effect of uridine on the rate of 5FU transport into Walker 256 tumor cells.
Walker 256 cells were incubated with different concentrations of 5FU, (0.5,1.0 and
1.5 mM) and uridine (5,10 and 20 mM), simultaneously for 1-min at 37°C in
glucose and Na+ containing medium. The intracellular uptake of SFU into the cells
was measured at 1-min accumulation time as previously described. % Inhibition is
expressed in comparison with the uptake of SFU at the particular concentration in the
absence of uridine at the same accumulation time.
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■ N M M I 60.00
40.00
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20.00
wmm
0 10 20 30
[I], conc. of uridine (mM)
0.5 mM SFU
1.0 mM SFU
1.5 mM SFU
Figure 4.18: Effect of uridine on the rate of 5FU transport into Walker 256 tumor
cells. Walker 256 cells were incubated with different concentrations of 5FU, (0.5,1.0
and 1.5 mM) and uridine (5, 10 and 20 mM), simultaneously for 1-min at 37°C in
glucose and Na+ containing medium. The intracellular uptake of 5FU into the cells
was measured at 1-min accumulation time as previously described. % Inhibition is
expressed in comparison with the uptake of SFU at the particular concentration in the
absence of uridine at the same accumulation time.
95
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Analysis of these results using the Dixon plot (94) to determine whether the
inhibition is competitive or non-competitive at multiple concentrations of both the
inhibitor (I), uridine and the substrate (S), 5FU, show evidence of competitive
inhibition. Figure 4.19 is the Dixon plot (1/u versus I), which is a plot of the
reciprocals of the initial rates of 5FU influx into the cells at varying concentrations
of 5FU against different concentrations of uridine, acting as an inhibitor of 5FU
influx. The resultant series of straight lines for different concentrations of 5FU
intersected at the point where the inhibitor concentration on the x-axis is equal to the
negative value of the Kj value, the inhibition constant. The intersection of the lines
above the x-axis in the Dixon plot is consistent with simple competitive inhibition
with an apparent Kj value of 10-mM (94).
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0 0 ^ 5
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0 03
0 03C
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0.5 mM 5FU
_ 1.0 mM SFU
1.5 mM 5FU
[I], Cone, of uridine (mM)
Fig. 4.19: Dixon plot of the competitive inhibition of 5FU transport by uridine.
Walker 256 cells were incubated with different concentrations of 5FU (0.5, 1.0 and
1.5 mM) and different concentrations of uridine (5,10 and 20 mM) simultaneously
for 1-min at 37°C in glucose and Na+ containing medium. Intracellular uptake of
5FU into the ASF was measured as previously described. The points at 0 represent
the uptake of SFU into the cells at the different concentrations of 5FU in the absence
of uridine. Apparent K j = 10 mM.
97
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Analysis of the results using the Comish-Bowden method (95), a plot of s/o versus I,
gave parallel lines with no intersection (Fig. 4.20), which is also consistent of
competitive inhibition. In this plot, S is the substrate (5FU) concentration, u is the
initial rate of influx at each concentration of the substrate, and I is the concentration
of the inhibitor (uridine).
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[I], conc. of uridine (m M )
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1.0 mM SFU
* 1.5 mM SFU
Fig. 4.20: Comish-Bowden plot of competitive inhibition of 5FU transport by
uridine.
98
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4.11 EFFECT OF DIPYRIDAMOLE ON SFU TRANSPORT
The initial transport of 5FU (1 mM) into the Walker 256 cells was measured in the
presence of different concentrations of dipyridamole, an inhibitor of the nucleoside
transport system. The transport of 5FU into these cells was significantly inhibited by
dipyridamole at the different concentrations tested. At 5 mM concentration of
dypridamole, the intracellular transport of SFU (1 mM) within the first minute of
incubation was inhibited by almost 50%. At 20 mM concentration of dipyridamole,
the intracellular transport of SFU (1 mM) was inhibited by greater than 60% (Table
4.10).
Figure 4.20, shows a plot of the reciprocals of the initial rates of 5FU influx into the
Walker 256 cells, in the presence of different concentrations of dipyridamole.
Figure 22 is a representative spectrum of intracellular 5FU measured in the ASF of
the Walker 256 cells exposed to 1 mM 5FU in the presence of 20-mM dipyridamole.
The 5FU signal measured at a chemical shift of about 34.7-ppm shows a significant
inhibition of the 5FU transported intracellularly.
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Compounds
Intracellular SFU
(pg/mg protein/min) % Inhibition
5FU alone 88.4 +.6.5
5FU + 5 mM DPD 44.8 ± 4.0 49.32
5FU + 10 mM DPD 40.4 ±2.8 54.30
5FU + 20 mM DPD 34.1 ±2.4 61.43
Table 4.10: Effect of dipyridamole (DPD) on the transport of SFU.
Walker 256 cells were incubated with 1 mM 5FU and indicated concentrations of
dipyridamole simultaneously in glucose and Na+ containing medium at 37°C for 1
min. The accumulation of free 5FU into the Walker 256 cells was measured in the
ASF as previously described. % Inhibition is expressed in comparison with the
uptake of 5FU in the absence of dipyridamole at 1-min accumulation time.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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0 10 20 30
[I] Dipyridamole, m M
Figure 4.21: Reciprocals of the initial rates of 5FU transport against different
concentrations of dipyridamole.
Walker 256 cells were incubated with 1 mM 5FU and indicated concentrations of
dipyridamole (5, 10 and 20 mM) simultaneously in glucose and Na+ containing
medium for 1 minute.
101
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Figure 4.22: A representative 1 9 F spectrum of the ASF of cells exposed to 1 mM SFU
in the presence of 20 mM dypridamole.
102
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4.12 EFFECT OF NITROBENZYLTHIOINOSINE (NBTI) ON SFU
TRANSPORT.
The effect of NBTI, a more specific nucleoside transport inhibitor, on the transport
of 5FU (1 mM) into the Walker 256 cells was studied by measuring the transport of
5FU into the cells in the presence of 1 pM concentration ofNBTI. At 1 pM
concentration ofNBTI, the intracellular transport 5FU (1 mM) into the Walker 256
cells proceeded unhindered (Table 4.11). There was no significant difference
between the intracellular transport of 5FU into the Walker 256 cells in the presence
or absence of 1 pM concentration ofNBTI.
Intracellular SFU
(pg/mg protein/min)
% Inhibition
SFU (1 mM) 88.4 ±6.5 -
5FU (1 mM) + 1 pM
NBTI
83.7 ±5.0 5.28
Table 4.11: Effect of Nitrobenzylthioinosine (NBTI) on SFU Transport into Walker
256 tumor cells. Walker 256 cells were incubated with 1 mM SFU and 1 pM NBTI
simultaneously in glucose and Na+-containing medium for 1-min. Intracellular
accumulation of SFU into the cells under this condition was compared to uptake in
the absence ofNBTI.
103
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4.13 EFFECT OF MODULATORS ON SFU TRANSPORT INTO TUMOR
CELLS:
4.13.1 Enhancement o f SFU transport by Methotrexate (MTX):
The effect of MTX as a modulator of SFU transport into the Walker 256 cells was
studied by measuring the transport of 1 mM 5FU into the Walker 256 cells after pre
incubating the cells with 5 mM MTX for varying lengths of time (10, 20, 30 and 60
min). After a 30-minute pre-incubation of cells with 5 mM MTX, the intracellular
transport of 1 mM SFU was enhanced by 26% within the first minute of incubation
with 5FU (Table 4.12).
After 60-minutes of pre-incubation with 5 mM MTX, the intracellular transport of
SFU was increased by almost 34% within the first minute of incubation with 1 mM
5FU (Table 4.12). The anabolism of 5FU to its fluronucleosides was enhanced by
pre-incubating cells with 5 mM MTX for 20-min prior to the introduction of 5FU.
There were detectable traces of fluronucleosides of 5FU measured at a chemical shift
of 39.30 ppm, 3 - 4 ppm upfield from 5FU within just the first minute of incubation
of the cells with SFU after the cells were pre-incubated with 5 mM MTX (Fig. 4.2).
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Length of pre-incubation
(min)
Intracellular SFU (pg/mg
protein/min)
% Increase
0 88.4 ±6.5 - •
10 73.19 ±8.2 -
20 93.59 ±8.8 5.87
30 111.63 ±9.7 26.28
60 118.40 ±8.2 33.93
Table 4.12: Enhancement of 5FU Transport by Methotrexate.
Walker 256 cells were pre-incubated with 5 mM MTX for indicated lengths of time.
1 mM SFU was added to the incubation medium and incubated with cells for 1-min.
The accumulation of 5FU in the cells was measured after 1-min of exposure to pre
treated cells.
% Increase is expressed in comparison with the uptake of 5FU into the cells at the
same accumulation time without pre-incubated with MTX.
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120
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0-min 10-mins 20-mins 30-mins 60-mins
Length of Pre-incubation
Figure 4.23: Effect of MTX on the Transport of 5FU into Walker 256 cells.
Walker 256 cells were pre-incubated with 5 mM MTX for indicated lengths of time
in glucose and Na+ containing bicarbonate Ringer’s buffer. 1 mM 5FU was added to
the incubation medium and incubated with cells for 1-min. The accumulation of 5FU
in the cells was measured in the ASF after 1-min of exposure to pre-treated cells.
106
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Figure 4.24: A representative 1 9 F spectrum of the ASF of cells exposed to 5FU after
20-min pre-incubation with MTX.
Walker 256 cells were pre-incubated with 5 mM MTX for 20-min in glucose and
Na+ containing bicarbonate Ringer’s buffer. 1 mM 5FU was added into the
incubation medium and incubated with cells for 1-min. The intracellular
accumulation of 5FU at 1-min accumulation time was measured in the ASF
previously described. The 1 9 F NMR spectrum was obtained using the following
parameters: SW = 14,992.5, PW = 26, NT = 28,000, D1 = 5-secs. The 5FU peak was
measured at 34.7 ppm. The fluoronucleosides were measured at a chemical shift of
39.30 ppm. The fluoronucleosides peak is phased in the opposite direction as the
5FU peak.
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Figure 4.25: A representative 1 9 F spectrum of the ASF of cells exposed to 5FU after
60-min pre-incubation with MTX.
Walker 256 cells were pre-incubated with 5 mM MTX for 60-min in glucose and
Na+ containing bicarbonate Ringer’s buffer. 1 mM 5FU was added into the
incubation medium and incubated with cells for 1-min. The intracellular
accumulation of 5FU at 1-min accumulation time was measured in the ASF
previously described. The 1 9 F NMR spectrum was obtained using the following
parameters: SW = 14,992.5, PW = 26, NT = 28,000, D1 = 5-sec. The 5FU peak was
measured at a chemical 34.75 ppm. Detectable traces of the fluoronucleosides were
lost in the phasing.
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CHAPTER V
5.0 DISCUSSION
5.1 BIOLOGICAL MODELS
5.1.1 WALKER 256 ADENOCARCINOMA
Walker 256 adenocarcinoma is an epithelial rat breast carcinoma that was established
in March, 1954 by R. N. Hull, W. R. Cherry, and I. S. Johnson from tumor tissue of a
Walker rat carcinoma maintained in adult Harlan-Wistar rats. Presently, the Walker
256 cells are cultured in 95% Medium 199 and 5% horse serum. An inoculum of 105
viable cells/ml in the above culture medium at 37°C, in an atmosphere of 5% carbon-
dioxide-95% air, increases approximately 6- to 10-fold in 5-7 days.
Being a breast cancer cell line, Walker 256 cells therefore present a reasonable model
to study the transport of 5FU across tumor cell membranes. In addition, this cell line
appears to have a slow metabolism of 5FU, making it possible to study the transport
of 5FU across the cell membranes without complications from extensive metabolism.
Prior studies in this laboratory have utilized the Walker 256 cells to study the
tumoral pharmacokinetics and metabolism of 5FU (31). This tumor model has also
been used in the study of nucleoside transport (74, 96). These authors also studied
109
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the nature and characteristics of the transporters expressed on the Walker 256 cells
(74, 96).
5.1.2 SIGNIFICANCE OF THE ANIMAL MODELS
Studies were initiated in animal models (female Sprague Dawley rats) bearing the
Walker 256 adenocarcinoma to understand the linearity of 5FU transport into tumor
cells and the dose dependence of the transport process. Hence, is the transport of 5FU
into tumor cells a saturable process, especially within the concentrations seen by
tumors in vivo? Though tumor models in rats differ to a good degree from tumors in
humans, the information obtained from tumors in rats could form a building block
upon which to extrapolate and understand what occurs in humans.
In comparison with the isolated cell culture system, the use of animals in these
studies presents a reasonable model because the living system retains the full
structural complexity of the tumor in vivo especially, the presence of the interstitial
fluid space. The role of the tumor interstitial fluid space in the transport of anticancer
drugs into the tumor cells is becoming increasingly important. Therefore, studies
with the animal models would make it possible to understand the impact of the
interstitial fluid space on 5FU transport into the tumor cells.
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In studies by Muller, et al, on the kinetics of 5FU in the interstitial fluid space, a high
interstitial tumor area under the curve (AUC) was associated with increased tumor
response to 5FU, whereas they found no association between tumor response and
plasma AUC (97). This observation suggests that the kinetics of 5FU in the
interstitial fluid space could be more predictive of response rather than the plasma
kinetics. It has been suggested that the tumor interstitial fluid space may also affect
the ultimate transport of drug molecules into the tumor cells (98).
The composition of the interstitial fluid space of neoplastic tissues is significantly
different from that of normal tissues. It is characterized by large interstitial space,
high collagen concentration, low proteoglycan and hyaluronate, high interstitial fluid
pressure and flow, absence of anatomically well-defined functional lymphatic
network, and high effective interstitial diffusion coefficient of macromolecules (98).
In as much as these characteristics favor the movement of macromolecules in the
tumor interstitium, high interstitial pressure and low microvascular pressure may
actually hinder the efflux of molecules from the interstitial fluid space into the tumor
cells. This may also explain why several anticancer agents fail to exert sufficient
cytotoxicity against solid tumors in vivo despite effective inhibition of tumor growth
in vitro. Therefore, with the animal models, it was possibly though indirectly, to
evaluate the impact of the interstitial fluid space on the transport of 5FU into the
tumor cells.
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5.1.3 SIGNIFICANCE OF THE CELL CULTURE SYSTEM
Although the animal models mimic more closely the intact tumor in humans, they do
not permit a direct study of the processes that regulate the transport of 5FU into the
tumor cells. Cell cultures have primarily been used to study the transport of
substances from the extracellular compartment into the intracellular compartment.
Nucleoside and nucleobase transports systems have been studied using cell cultures
of different cell types (For a review, see ref. 63). Therefore, after generating initial
data using the animal models, cell cultures were utilized for subsequent studies
because they allow a manipulation of the processes and allow the study of one
process at a time. With the cell cultures, it was possible to study the impact of several
agents on the transport of 5FU and the kinetics of the transport from the extracellular
space into the intracellular space.
In addition, the cell culture system made it possible to answer some of the questions
that we were asking on the transport of 5FU such as:
(i) What is the rate of transfer?
(ii) What factors govern this rate of transfer?
(iii) Is the transport of 5FU into tumor cells an active transport process or is by
simple diffusion?
(iv) Is the transport Na+ dependent?
112
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What agents could be used to enhance or inhibit transport of 5FU into the tumor or
non-tumor cells respectively?
The answers to some of these questions could not only help in understanding the
mechanism of transport of 5FU but also assist in the manipulation of this process for
optimizing cancer treatment with 5FU. These questions and more could only be
addressed and answered using the isolated cell culture system.
One technique that appeared particularly attractive in answering these questions
using an isolated system is the use of tumor spheroids grown in NMR tubes. This
technique has been used on studies of 5FU and other drugs under non-invasive
dynamic conditions (91, 99-102).
Multicellular tumor spheroids are spherical aggregates of malignant cells. They
mimic tumors in vivo by showing an external layer of proliferating cells, an
intermediate layer of quiescent cells and an internal layer of necrotic cells. As a
result of these characteristics, they serve as good models of poorly vascularized
tumor tissues. A number of studies have documented the ability to use tumor
spheroids in carrying out dynamic, real time studies using NMR spectroscopy (99,
101,102). These studies also demonstrated the possibility of growing spheroids
within the NMR tube under conditions that closely simulate normal growth
conditions (99). In light of using the NMR technique as the tool of analysis, the
113
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tumor spheroids provide an added advantage of increasing the signal-to-noise ratio
by making it possible to introduce a large number of cells into the NMR tube.
Despite these advantages of using the tumor spheroids to perform studies on the
transport of 5FU into tumor cells, this technique has the limitation of spheroids
adhering to each other inside the NMR tube and to the walls of the NMR tube. This
phenomeum causes poor perfusion and introduces another level of complexity. This
and the complicated setup process are time consuming. As a result, the studies herein
were initiated and performed using monolayer cell cultures. Under the experimental
conditions employed in these studies, it is possible to extrapolate these results and
gain an understanding of the in vivo processes.
The monolayer cell culture has the advantage of allowing 5FU to gain direct access
to the cells thereby permitting the measurement of one process at a time. It however,
has its share of complexity. While doing these studies, we observed that the
generation of cells used for the studies affected the uptake of 5FU into the cells.
Later passages of cells transported 5FU to a much lesser degree than did the earlier
passages. It is possible that as the cells were routinely sub-cultured from one passage
to another, they tended to loss some of their morphological characteristics resulting
in the loose of some of the transporters on the cell membranes that permit the entry
of substances into the cells.
114
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Based on these observations, the studies reported here were done with cultures that
where no more than 4 passages apart from the original cells purchased from ATCC.
We also observed that the degree of confluence of the monolayer cells also affected
the transport of 5FU into the cells. Confluent cells took up 5FU to a much lower
degree than did cells that were not completely confluent. Tumor cells are known to
be very rapidly dividing, as are gastrointestinal tissues. Hence, these cells exhibit
higher transport efficiency and have the ability to take up exogenous substances
more rapidly than do normal cells. However, confluent cells are dormant and are not
as rapidly dividing as non-confluent tumor cells. Hence, they would not take up
compounds from the extracellular environment as rapidly as would non-confluent
tumor cells. Therefore, the degree of confluence of the cells used for the transport
studies was a very important consideration in carrying these studies, and nearly
confluent cells were used for all the studies. The degree of confluence was
determined by visual inspection and maintained at approximately 90%. The selected
level of confluence was balanced against the desire to achieve a high density of cells
in order to enhance the detectability of fluorinated signals using the 1 9 F NMR
Spectroscopy.
Studies with cell cultures require a close attention to all experimental details, as little
deviations could have a large impact on the efficiency of 5FU transport into the cells.
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When cells were exposed to 5FU without prior incubation for at least 1 hr in serum
free medium, 5FU was transported very poorly into the tumor cells (Fig. 5.1).
200.0
u.
150.0
Pre
incubated
No pre
incubation
100.0
50.0
0.0
Time (mins)
Fig. 5.1: Effect of pre-incubation in serum free buffer on the transport of 5FU.
Figure 5.1 shows the transport of 5FU (1 mM) into the Walker 256 cells without
prior incubation of cells in serum free medium for at least 1 hour. This is compared
to 5FU transport into the cells after the cells were washed and pre-incubated in serum
free medium 199 for 1 hr at the same accumulation times. The intracellular
accumulation of 5FU at the same time points, show the efficiency of 5FU transport
was higher when cells were washed and pre-incubated in serum free medium. It is
possible that the pre-incubation of cells in serum free medium prior to exposure of
the cells to 5FU caused the depletion of endogenous nucleosides and nucleobases
making the cells more apt to take up 5FU. It is also possible that pre-incubation in
serum free medium, depleted most of the nucleosides and nucleobases on the cell
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surface from the serum that could otherwise compete for uptake with 5FU. In any
case, pre-incubation of the cells in serum free medium resulted in a higher uptake of
5FU into the tumor cells and was routinely done in all studies.
5.2 DETECTION OF PRODUCTS
5.2.1 1 9 F NMR SPECTROSCOPY
1 9 F NMR spectroscopy has been used as the mode of analysis in these studies, to
detect and quantitate the 5FU transported into the tumors and other tissues of
interest. The nuclear magnetic resonance technique provides a unique approach to
study intracellular metabolic processes. Since the intracellular metabolism of a
substance is determined by the degree of its intracellular uptake, this technique can
and has been successfully utilized to study the transport of 5FU from the
extracellular space into the intracellular compartment of the cell.
In this study, it permitted the direct and simultaneous identification and quantitation
of all fluorinated compounds present at the tumor site and was able to distinguish
between and quantify relative amounts of specific metabolites in the intact cell,
provided these compounds were present in high enough concentrations. Studies
using NMR spectroscopy to assess the intracellular uptake of 5FU, revealed
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information on the relative amounts of the nuclei in different environments as
determined from the signal intensities for resonances at different chemical shifts.
The NMR technique is inherently insensitive. Due to this inherent insensitivity,
higher concentrations of 5FU were used for these studies to increase the signal to
noise ratio and allow a better detection of any fluorinated signals. Also, as a result of
the poor sensitivity, many free induction decays (FIDs) had to be accumulated on
each sample, making data acquisition times to be very long. Primarily, an increase in
the magnetic field strength is exploited as a means to increase the sensitivity of the
magnet and increase the signal-to-noise (S/N) ratio of a sample. Therefore, in these
experiments, the power of the 500 MHz magnet with a higher magnetic field strength
to increase the detectability of the signals was tested. However, when compared to
the 200 MHz magnet, there was no increase in the S/N ratio between the two
magnets. For 1 9 F NMR studies, the lower magnetic field strength magnet provided a
better S/N ratio than the 500 MHz.
Another limitation of this technique is that since it requires the presence of a fluorine
atom as an integral part of the compound under study, it was not possible to measure
the transport of non-fluorinated compounds, since they lacked a fluorine atom. For
instance, in the inhibition studies utilizing uridine as an inhibitor of 5FU transport, it
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would have been of interest to also measure the transport of uridine in the presence
of 5FU.
5.3 THE TRANSPORT OF 5FU INTO WALKER 256 TUMOR CELLS AS AN
ACTIVE TRANSPORT PROCESS
This section of the discussion analyzes what we have learnt in our attempt at
understanding the mechanism and regulation of transport of 5FU into tumor cells
using the Walker 256 adenocarcinoma cells as the tumor model. There exists a
controversy regarding the nature of 5FU transport into tumor cells, whether transport
occurs by an active transport (50), facilitated transport (48, 49, 103) or by simple
diffusion (48) and whether the transport is dependent (40, 41, 45) or independent of
Na+(50, 51).
The plasma t1 / 2 of 5FU has been shown to be about 10 - 20 min (13), while free 5FU
has been observed in the tumor hours after it has cleared the blood (1, 2,13). This
long tumoral half-life has been associated with the “trapping” of free 5FU in the
tumor. The question then has been, what is the mechanism that determines trapping?
In as much as free 5FU is the species that is trapped, trapping appears to be a
transport based phenomena. We present evidence that the process of transfer of 5FU
from the tumor interstitial fluid space into the tumor cells is a saturable and an active
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transport process that is regulated by the presence or absence of transport proteins on
the tumor cell membranes.
These studies were initiated in animal models (female Sprague Dawley rats) to first
understand the linearity of 5FU transport into tumor cells and the dose dependence of
the transport process. In essence, is the transport of 5FU into tumor cells a saturable
process, especially within the concentration ranges seen by the tumors in vivo?
In the tumor acid soluble fraction, free 5FU was observed as the major component 2
hours post drug administration (Figure 4.1). The amount of 5FU measured in the rat
tumors, following administration of two widely different doses (30 mg/kg and 150
mg/kg) of 5FU, was consistent with a non-linear relationship between the
administered dose of 5FU and its intracellular uptake. A five-fold increase in
available (administered dose) 5FU translated itself to only a 50% increase in
intracellular 5FU measured in the tumor (Table 4.1), suggesting a saturability of the
transport process.
On the other hand, in a tissue such as the liver, where the uptake of 5FU was more
likely to be metabolically-driven rather than transport-driven, the amount of free 5FU
detected at 2 hours post drug administration was barely above background (Figure
4.2). a-fluoro-p-alanine (FBAL), a major catabolite of 5FU remained the major
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component of the liver ASF after 2 hours of drug administration (Figure 4.2). These
results are supportive of the suggestion that the transport of 5FU from the interstitial
fluid space into the tumor cells is a saturable active transport process.
The transport of 5FU, which occurs primarily at the cellular level, can be described
by a schematic diagram below describing the fate of 5FU upon systemic
administration.
Transport Phase Cell Uptake Phase Metabolic Phase
5-FU
5-FU
FNUC
in systemic
in tumor
FUR, FUdR,
biood pool
cell space
FUMP, etc
i
t
1
5-FU
5-FU in
F-RNA
in tumoral
interstitial
5FU-TS
blood pool
fluid space
F-DNA
Wolf et al (6)
The transport phase describes the uptake of 5FU from the site of administration into
the systemic blood pool and from the systemic blood pool into the tumoral blood
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pool. The process of transfer from the systemic blood pool to the tumoral blood pool,
depends to a large extent on the flow of blood to the tumor and on the perfusion of
the tumor. As previously stated, tumors in vivo have varying perfusion, therefore the
degree of perfusion of the tumor would determine to a large extent, the amount of the
administered drug that eventually makes it into the tumoral blood pool.
The cell uptake phase describes the ability of the drug in the tumoral blood pool to
diffuse into the tumor interstitial fluid space and then get transported into the tumor
cells. The transfer process from the tumoral blood pool to the interstitial fluid space
is largely by simple diffusion, and is affected by the osmotic pressure of the
interstitial fluid space (98). From the interstitial fluid space, 5FU gets transported
into the tumor cells. Once inside the tumor cells, 5FU undergoes metabolism into
active anabolites bringing it into the metabolic phase. This phase describes the
activation of 5FU into fluoronucleotides and fluoronucleosides and their subsequent
incorporation into RNA, DNA and inhibition of thymidylate synthase.
The fact that free 5FU remained in the tumor as the major component 2 hours post
drug administration, similar to observations in human tumors, suggest that 5FU
transport into the tumor is not metabolically-driven as initially thought but suggests
the presence of a carrier-mediated active transport process.
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What is most interesting, however, is the striking similarity in the pattern of the
fluorinated pyrimidines measured in these tumor cells, whether they were grown in
cell culture or in the flank of a rat. Free 5FU was the major intracellular
fluoropyrimidine present in both cases (Figures 4.1, 4.4 & 4.5). The conversion of
5FU into its anabolites: the 5-fluorouridine and 5 -fluorodexoyuridine, the
ribonucleotides, the deoxyribonucleotides, appears to be a relatively slow process in
these cells.
5FU taken up into tumor cells has been shown to undergo metabolism into active
fluoronucleotides and nucleosides such as fluorouridine 5’-triphosphate (FUTP) and
5 -fluoro-2 ’ -deoxyuridine- 5 ’ monophosphate (FdUMP) (14, 19) and subsequent
incorporation into RNA and inhibition of thymidine synthase upon incorporation into
tumor cells. Therefore, it was important to study the transport of 5FU into tumor
cells in the absence of extensive intracellular metabolism.
Under the experimental conditions employed in the cell culture studies, most of the
5FU transported into the tumor cells remained as free 5FU and accumulated in the
acid soluble fraction (ASF) (Figures 4.4, 4.5, 4.6 and 4.7). There were no detectable
metabolites even after 60 minutes of incubation. These results compare well with
those seen in Ehrlich Ascites cells (50) and Lettre cells (51).
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The following results from the cell culture experiments also support the presence of a
carrier-mediated active transport of 5FU into Walker 256 cells.
(I) Free 5FU accumulated against its concentration gradient in the Walker 256
cells after 90-min of incubation, accounting to almost 3.5 times the
extracellular concentration as measured in the acid soluble fraction (Fig. 4.8)
within 10-min of incubation.
(II) The transport of 5FU was inhibited in the absence of extracellular glucose
(Table 4.4) and in the presence of a glycolytic inhibitor, iodoacetate (Table
4.5), in separate experiments.
(III) Structurally related analogs, uracil, thymine, uridine and cytidine
significantly inhibited the transport of 5FU into the cells (Table 4.8).
The transport of 5FU into the Walker 256 cells against its concentration gradient
(Fig. 4.8) indicates an active transport process. The concentration of 5FU measured
intracellularly equilibrated and exceeded the concentration of 5FU in the
extracellular medium within 1-min of incubation of cells with 5FU, showing a 2:1
ratio of 5FUin t /e x t by 1-min incubation. With increased incubation time, 5FU
continued to accumulate intracellularly against its concentration gradient accounting
to 3.5 times the concentration of 5FU in the incubation medium within 10-min of
incubation. These results compare well with those of Ojugo (51) in Lettre cells where
a maximum of 3:1 int/ext 5FU ratio was recorded at 0.1 mM 5FU and studies in
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Ehrlich Ascites tumor cells by Yamamoto (50) reported a maximum of 3:1 int/ext
5FU ratio at 0.8 mM 5FU.
The active transport of 5FU into the Walker 256 cells showed a time dependent
transport process with a rapid initial uptake (@ 1 min incubation time) (Fig.4.7) and
equilibration of transport at 10 minutes of incubation. Further incubation beyond 10
min showed no increase in intracellular accumulation of free 5FU measured in the
acid soluble fraction, though the intracellular concentration remained above the
extracellular concentration for the 90-min of incubation.
The fact that the intracellular accumulation of 5FU increased for up to 10-min after
incubation with no further increase even with continued incubation but rather a
decrease in the intracellular free 5FU could be the result of two processes: the efflux
of 5FU from the tumor and/or its metabolism. Though 1 9 F NMR spectroscopy is able
to give exquisite chemical information of the fluorinated species present,
distinguishing between the various species present, it has an inherently poor
sensitivity and may not be able to detect low metabolite levels. The generated
nucleosides though below NMR detectable limits could exit the cells and potentially
compete for uptake with 5FU, thereby decreasing the amount of free 5FU measured
intracellularly.
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The inhibition of 5FU transport in the absence of extracellular glucose and by a
glycolytic inhibitor, iodoacetate indicates a dependence of the transport process on
energy obtained through glycolysis. In addition, the finding that the uncoupling agent
2,4-dinitrophenol which inhibits the synthesis of ATP in the mitochondria failed to
inhibit the transport of 5FU, support the finding that the active transport of 5FU in
these cells is primarily dependent on energy obtained through glycolysis.
Active transport of a substance involves the transport of that substance against its
electrochemical gradient and is usually coupled to a source of energy. Most primary
active transport processes in mammalian cells are coupled to cellular ATP as a
source of energy. ATP required to drive an active transport can be obtained through
different ways. Synthesis of ATP in the mitochondria is one way and generation of
ATP from glycolysis is another source of ATP required to drive a primary active
transport process, though this pathway generates a lower percentage of ATP when
compared to ATP synthesized in the mitochondria. In these experiments, it appears
that the lower amount of ATP generated from glycolysis was sufficient to drive the
active transport of 5FU.
It can be noted that though the intracellular accumulation of 5FU was inhibited by
the absence of extracellular glucose (36%) and by iodoacetate, a glycolytic inhibitor
(44%), the inhibition appeared to be partial. This finding could suggest the presence
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of another transport mechanism for 5FU into these cells that may not require an
energy source. It is possible that there is another carrier-mediated transport that is not
energy dependent.
Pyrimidine nucleobase analogs, (uracil and thymine) and nucleoside analogs (uridine
and cytidine) significantly inhibited the transport of 5FU (Table 4.8), suggesting the
presence of a common transport carrier that mediates their uptake into the tumor
cells. Uracil inhibited the transport of 5FU by only 11% possibly because of its poor
solubility in the incubation medium hence, decreased amount of uracil available for
competition with 5FU. Cytosine failed to inhibit 5FU transport into these cells
because the mode of uptake of cytosine is by passive diffusion (44). Thymine
inhibited 5FU transport by 34% suggesting a common carrier between the two
analogs. The fact that uridine inhibited 5FU transport by almost 50%, much higher
than the inhibition observed by nucleobase analogs (uracil and thymine) suggest the
presence of a shared transport system between nucleosides and nucleobases in these
cells and more so, that 5FU transport could be mediated by a nucleoside transporter.
Wohlhueter et. al (1980), reported similar findings in Novikoff hepatoma cells,
where nucleosides inhibited uracil and 5-fluorouracil transport more strongly than
the pyrimidine bases inhibited one another’s transport (49). In a different study in
Novikoff hepatoma cells on hypoxanthine transport, Plagemann and Wohlhueter
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found that nucleosides strongly inhibited the transport of hypoxanthine and vice-
versa (70, 72) in a competitive manner. This finding that nucleosides inhibit
pyrimidine nucleobase transport more strongly than do nucleobases themselves
suggest that the effect is not an indirect one possibly from phosphorolysis of the
nucleosides (49) but as a direct evidence that they share the same transport system.
Interestingly, the intracellular transport of 5FU was enhanced at a lower extracellular
pH environment (pH 6.7), possibly because of the induction of a more negative pH
gradient across the tumor membrane (Table III). At extracellular pH 6.7, we
observed a 6:1 ratio of 5FUj n t /e x t whereas at extracellular pH 7.2, a 3.7: 1 ratio was
recorded at the same accumulation time (Fig. 5.2).
Figure 5.2: Ratio of intracellular/extracellular 5FU as a function of the extracellular
§ § 5FUint/ext
7.00
pH 7.2 pH 6.7
Buffer pH
pH
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The pH effect has been proposed as a mechanism that influences the uptake,
retention and elimination of 5FU from tumor cells (51, 52). An I 9 F MRS study by
Guerquin-Kem et al (1991) showed in a rat fibrosarcoma that the t1 / 2 of 5FU
elimination was 2.5-folcj^onger at intracellular (pH; ) pH < 6.9 than at pH; 7.3 (52).
Similarly, Ojugo et al (1998), reported in Lettre cells, that an increase in the -ApH
gradient across the tumor cell membrane correlated with an increase in the uptake of
5FU (5FUin /5FUe x t ratio) by the cells (51). At ApH = 0, 5FUi n t /e x t ratio was 1:1, at
ApH - -1, a > 1.5:1 ratio of 5FUin t /e x t was observed.
With a pKa of 8.1, about 90-95% of 5FU would be in the uncharged form at around
extracellular pH (pHe ) of 6.5-7.0. At pHe 7.2 and pHe 6.7, different amounts of 5FU
(in the uncharged form) would be available to be transported, with the higher amount
of free 5FU at pHe 6.7. This difference in available 5FU, the pKa of 5FU alone and
the distribution of 5FU as a weak acid alone cannot explain our findings in this
study. Our results are explained by a difference in the negative pH gradient across
the tumor cell membrane. Tumor intracellular pH is around 7.0 - 7.2. It means that at
pHe 7.2, the pH gradient was near zero. Even at -ApH = 0, 5FU was transported
intracellularly, more so against its concentration gradient. At pHe 6.7, a more
negative pH gradient was imposed across the tumor cell membrane enhancing the
drive of 5FU into the tumor.
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An alternative explanation to these findings is that co-transport of 5FU may occur on
a proton symport using the proton motive force of the change in pH gradient (ApH)
as a result of the electrochemical gradient of H+ that was imposed across the tumor
cell membrane.
The pH gradient of tumors in vivo is the reverse of that in normal tissues. In tumors,
the intracellular pH is alkaline and the extracellular pH is acidic probably because of
the high glycolytic rate associated with tumors and the subsequent extrusion of
cellular acids. Although the increased negative pH gradient across the tumor cell
membrane enhanced the transport of 5FU into the tumor cells, the pH effect is not
the only mechanism by which 5FU is transported and trapped in tumor cells. We
have shown transport of 5FU against its concentration gradient (Fig. 4.7 and 4.8) and
trapping of free 5FU within the tumor cells at a neutral pH environment (pH 7.2) and
zero pH gradient (Fig. 4.4 and 4.5).
In many animal cells, Na+ is needed to create the electrochemical gradient necessary
for the active transport of glucose and certain amino acids into the cell resulting in a
Na+ substrate co-transport. While the initial measurements had suggested a Na+
dependent transport process, a more complete kinetic study here have shown that
there is no significant difference in uptake between kinetic studies in the presence or
absence of extracellular Na+ (Fig. 4.9). Though the absence of extracellular Na+
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slightly inhibited the initial rate (@ 1 min incubation time) of uptake (Fig. 4.9), the
inhibitory effect disappeared with increased incubation and 5FU still accumulated
intracellularly against its concentration gradient even in the absence of extracellular
Na+ (Fig. 4.12). It is possible that increased incubation resulted in an equilibration of
Na+ across the membrane.
Hence, Na+ here could be acting as a cofactor (50) in the stimulation of 5FU transport
and not as a driving force for 5FU active transport into the cells, as occurs in the
active transport of glucose and many amino acids. While in the rat intestinal
preparations, a Na+ dependent nucleobase transporter have been reported (40, 41, 45),
in the few tumor cells examined, the transport of 5FU have been found to be
independent ofNa+ (50, 51). Further studies using ouabain, a Na+ K+ ATPase inhibitor
to abolish the Na+ gradient showed about the same degree of inhibition as observed
in the absence of extracellular Na+ at 1-min incubation time but had no effect on the
transport of 5FU at 10-mins incubation time (Table 4.3).
The transport of 5FU against its concentration gradient, the saturability (Km = 0.55
mM) of its active transport process across Walker 256 tumor cell membranes, in both
animal and cell culture studies, the dependence of the transport process on energy
from glycolysis, eliminates simple diffusion as the mode of uptake of 5FU into these
cells. Rather, it points to the presence of protein transporters on the cell surface that
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are mediating 5FU uptake into the cells. The question then becomes, what transporter
(s) are responsible for this uptake process?
In conclusion, the studies so far have shown that the transport of 5FU across the
Walker 256 tumor cell membranes is a saturable process active transport process
with a Km of 0.55 mM. The active transport of 5FU is independent of Na+ and
dependent on energy obtained through glycolysis. The transport was inhibited by
structurally related analogs, showing a common carrier between 5FU and the
different analogs. These studies have also shown evidence of the presence of a shared
transport system between pyrimidine nucleobases and nucleosides in the Walker 256
tumor cells evidenced by the more significant inhibition of 5FU transport by uridine.
5.4 MECHANISM OF THE ACTIVE TRANSPORT PROCESS OF 5FU INTO
THE WALKER 256 CELLS
We have previously shown the presence of a shared transport system between
nucleosides and nucleobases in the Walker 256 cells and the possibility that 5FU is
transported into these cells through a nucleoside transporter. With this understanding
of a carrier-mediated active transport of 5FU into Walker 256 tumor cells, this
section is focused at discussing the insights we gained into the nature of the
transporter (s) that is/are responsible for the translocation of 5FU, a pyrimidine
nucleobase into Walker 256 tumor cells. We also discuss the evidence that
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methotrexate (MTX) has the potential not only to enhance the anabolism of 5FU in
the tumors, but also affects the transport process and trapping of free 5FU in the
tumors.
The following results suggest the presence of an equilibrative transport mechanism
for 5FU into Walker 256 cells mediated through the ei nucleoside transporter:
(i) Uridine competitively inhibited the transport of 5FU into Walker 256 tumor cells.
(ii) Dipyridamole, a nucleoside transport inhibitor, significantly inhibited the
transport of 5FU into these cells.
(iii) The transport of 5FU into the Walker 256 cells was resistant to inhibition by 1
pM nitrobenzylthioinosine (NBTI).
This represents the first direct demonstration of 5FU transport into tumor cells via
the ei nucleoside transporter.
5FU transport was more strongly inhibited by uridine, a nucleoside analog than by
pyrimidine bases, uracil and thymine (Table 4.8). A more detailed investigation into
the nature and the kinetics of uridine inhibition of 5FU transport, show evidence of a
competitive inhibition of 5FU uptake into these cells by uridine (Figures 4.19 &
4.20). Analysis of the results with both the Dixon plot (Fig. 4.19) and the Comish-
Bowden plot (Fig. 4.20) showed evidence of a competitive inhibition between 5FU
and uridine.
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In the analysis of the results with the Dixon method (94), an increasing concentration
of 5FU in the incubation medium, caused uridine to become less inhibitory of 5FU
transport. The intersection of the lines above the x-axis is an evidence of competitive
inhibition. This means that the two substrates (5FU and uridine) bind to the same
position on the transporter. Therefore, as the concentration of 5FU whose transport
was being measured in this case was increased, there was more substrate available
for binding to that position on the transporter making uridine less inhibitory of 5FU
uptake. This finding confirms not just the presence of a shared transporter between
the two analogs, but that 5FU and uridine actually compete for the same site on the
transporter.
If on other hand, the competition between 5FU and uridine were non-competitive, in
which case the substrates would bind to different positions on the same transporter.
Therefore, increasing the concentration of 5FU in the incubation medium would not
affect the inhibitory effect of uridine as it would be equally effective at inhibiting the
transport of 5FU at the different concentrations of 5FU. In non-competitive
inhibition, the inhibitor binds at a different position on the same transporter as the
substrate. However, such binding of the inhibitor on the transporter at a different
position from the substrate affects the conformation of the transporter, which
ultimately affects the binding of the substrate to the transporter. There is no
competition for the same position on the transporter. In this case, the lines
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corresponding to the reciprocal of the initial rates of influx of 5FU at varying
concentrations of 5FU in the presence of different concentrations of uridine would
intersect on the x-axis (Dixon method) (94).
Though it would be desirable to also study and measure the transport of uridine as
the substrate in the presence of 5FU (as an inhibitor) as another means of
ascertaining competitive inhibition between 5FU and uridine, the tool of analysis for
this study (1 9 F NMR spectroscopy) makes that impossible. The idea would be to
check if 5FU equally inhibited uridine uptake in a competitive manner.
Investigating further the involvement of the nucleoside transporter in 5FU transport,
studies were done using dipyridamole, a nucleoside transport inhibitor to ascertain its
effect on 5FU transport. These studies on the effect of different concentrations of
dipyridamole on the initial influx of 5FU into Walker 256 cells show a significant
inhibition of 5FU transport by dipryridamole. At 5 mM concentration of
dipyridamole, the initial influx of 5FU was inhibited by almost 50% (Table 4.10).
Inhibition of 5FU transport by dipyridamole, further implicates the nucleoside
transporter and is consistent with the view that a nucleoside transporter mediates the
transport of 5FU into the Walker 256 cells. Studies by Plagemann and Wohlhueter
(1984), in Novikoff and HTC rat hepatoma, Chinese hamster ovary and Ehrlich
Ascites cells, found hypoxanthine transport to be inhibited by uridine and strongly
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inhibited by dipyridamole (IC5 0 = 100 to 400 nM) (70). These findings are in
agreement with the view that nucleosides and nucleobases share the same transport
system in some cell types such as the Walker 256 cells and more so that, 5FU
transport in these cells is mediated by the nucleoside transport system.
A number of studies that have demonstrated the involvement of both the es and ei
nucleoside transporters in the translocation of nucleobases in cultured mammalian
cells (63, 68, 72). In S49 cells, which express mainly the es transporter and lack the
purine nucleobase transport system, the flux ofhypoxanthine was observed to be
saturable and strongly inhibited by uridine, NBTI and dipyridamole. The efficiency
of transport was found to be low (68) when compared to cells that poses the purine
nucleobase carrier. Similarly, in Novikoff rat hepatoma cells and more recently in
ECV 304 cells, derived from human umbilical vein endothelial cells which posses
mainly the ei nucleoside transporter, hypoxanthine exhibited a high affinity for the ei
nucleoside transporter and the influx was inhibited by nucleosides (63, 72).
Even though dipyridamole significantly inhibited 5FU transport, there was still
intracellular transport of 5FU in the presence of 20 mM concentration of
dipyridamole. The non-complete inhibition of 5FU transport by dipyridamole, (Fig.
4.21) further suggest that there are more than one transport systems for 5FU in these
cells, facilitated transport and passive diffusion occurring concurrently. Several cell
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lines have been noted to have multiple transport systems for nucleosides and also for
nucleobases (63), concurrently transporting nucleosides and nucleobases into the
cells. For instance, in the Walker 256 cells, in addition to the presence of a facilitated
nucleoside transport involving the ei nucleoside transporter (74), a Na+ dependent
nucleoside transport has also been reported (96).
Studies on the effect of different concentrations ofNBTI, on the influx of 5FU into
Walker 256 cells show evidence of transport mediated through the ei nucleoside
transporter. 5FU transport into Walker 256 cells was resistant to inhibition by 1
micro-molar concentration ofNBTI, was only inhibited at a much higher
concentration ofNBTI (Table 4.11), implicating the NBTI-insensitive (ei) nucleoside
transporter for uptake of 5FU in these cells. Similarly, studies by Belt et al (71) have
documented that in Walker 256 cells, the initial rates of uridine, thymidine and
adenosine uptake were insensitive to 1 pM NBTI. These studies noted the absence of
high affinity NBTI binding sites in Walker 256 cells (74) and inhibition of the ei
nucleoside transporter only at a very high concentration (73).
Methotrexate was identified as an agent capable of enhancing the intracellular
transport of 5FU into tumors. Clinically, MTX is used in combination chemotherapy
with 5FU as a modulator of its anabolism to active fluoronucleotides and
fluoronucleosides (31-34). In these studies using Walker 256 cells in cultures, we
137
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have shown an increase in the intracellular transport of 5FU measured when cells
were pre-incubated with MTX. The degree of enhancement of intracellular transport
of 5FU was directly related to the length of pre-exposure to MTX (Figure 4.23). Pre
exposing the cells to 5-mM MTX for 20-mins showed only a 5% increase in the
intracellular uptake of 5FU (Table 4.12). Whereas, pre-exposure of cells to MTX for
60-mins before the introduction of 5FU resulted in a more than 30% increase in the
intracellular uptake of 5FU (Table 4.12).
These findings compare well with earlier studies by El-Tahtawy and Wolf, (31) in
rats which documented a significant decrease estimated at more than 3 orders of
magnitude in the elimination rate constant of 5FU when the rats had been pre-dosed
with MTX. By what mechanism does MTX enhance 5FU transport? It is possible
that MTX enhances the intracellular uptake of 5FU by blocking its efflux from the
tumors.
In conclusion, the studies here show that the transport of 5FU into Walker 256 tumor
cells was competitively inhibited by structurally related nucleoside analog, uridine,
significantly inhibited by dypridamole and resistant to inhibition by 1 pM
concentration ofNBTI. These findings are consistent with the view that nucleosides
and nucleobases share the same transport system in the Walker 256 cells and that
5FU transport is mediated through the ei nucleoside transporter. These studies have
138
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also documented the ability of MTX, not only to modulate the anabolism of 5FU but
also to enhance the transport of 5FU into tumor cells and its trapping in the cells.
This understanding could allow the identification of more effective modulators of
5FU transport into the tumor cells and allow a more rationale combination therapy
utilizing 5-Fluorouracil. This information could also be useful in the synthesis of
new chemotherapeutic agents that may target the nucleoside transporter for entry into
the tumor cells.
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CHAPTER VI
— -.......- -- - 6.0- - ...-.....- - - - SUMMARY - .................
There is no doubt that understanding the mechanism and regulation of the transport
of 5FU into tumor cells would enhance its therapeutic effectiveness by allowing the
identification of appropriate transport modulators in combination chemotherapy.
These studies in Walker 256 tumor cells have documented the presence of a saturable
(Km = 0.55 mM) carrier-mediated active transport of 5FU into tumor cells.
The transport of 5FU into the tumors of rats bearing the Walker 256 tumors
demonstrated a dose dependent uptake process and a non-linear relationship between
the administered dose of 5FU and its uptake into the tumors. These results are
suggestive of a saturable transport process. Free 5FU was measured as the major
component of the tumor 2 hours post drug administration. Interestingly, in the liver
free 5FU could not be trapped even after 2 hours of exposure to 5FU because the
process of uptake was metabolically driven rather than transport-driven.
In these cell culture studies, a saturable active transport process of 5FU in the Walker
256 tumor cells was documented. 5FU accumulated intracellularly against its
concentration gradient both in the presence and in the absence of extracellular Na+
140
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(Fig. 4.8 & 4.12). 5-mins after exposure of cells to 5FU, it equilibrated across the
intracellular and extracellular compartments, and continued to accumulate inside the
cells. This intracellular accumulation of 5FU against its concentration gradient is
indicative of an active transport process. The active transport of 5FU into these cells
was dependent on energy obtained through glycolysis as the transport was
significantly inhibited by the absence of extracellular glucose and by the presence of
iodoacetate, a glycolytic inhibitor.
The transport of 5FU into these cells was found to be dependent on the pH gradient
imposed across the tumor cell membranes. The more the negative pH gradient
(-ApH) imposed across the tumor cell membrane, the more 5FU accumulated inside
the tumor cells. At extracellular pH 6.7, the intracellular transport of 5FU was
increased by 38%, compared to the accumulation at extracellular pH 7.2. Though the
tumor pH gradient affected the uptake of 5FU, it is does not appear to be the only
mechanism that regulates the transport of 5FU into tumor cells.
Structurally related analogs, uracil, thymine, uridine and cytidine inhibited 5FU
transport into the cells by 11.5%, 34.4%, 48.9% and 53.4% respectively. This
suggests not just the presence of a carrier-mediated transport for 5FU but also the
presence of a common carrier between the analogs. The finding that the nucleoside
analogs uridine and cytidine, inhibited 5FU transport more significantly than did the
141
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nucleobase analogs suggest that 5FU is possibly transported into the cells through
one of the nucleoside transporters.
Kinetic studies on the inhibition of 5FU transport by uridine at various
concentrations of both 5FU and uridine, analyzed by both the Dixon and the Comish-
Bowden methods were consistent with a competitive inhibition, K j = 10 mM. These
results further point to the involvement of a nucleoside transporter in the
translocation of 5FU in these tumor cells. This view was supported by the significant
inhibition of 5FU transport by dypridamole, a nucleoside transport inhibitor. 5-mM
dypridamole inhibited the transport of 1 mM 5FU into these cells by almost 50%
within the first minute of incubation. On the other hand, the transport of 5FU was
resistant to inhibition by 1 pM NBTI and was only inhibited by 5 mM NBTI.
Methotrexate (MTX), was identified as a modulator of 5FU transport. Pre-incubation
of cells with MTX showed an enhancement in the intracellular transport of 5FU with
the degree of enhancement being dependent on the length of pre-incubation with
MTX. After 60-mins of pre-incubating cells with 5 mM MTX, the intracellular
transport of 5FU (at 1-min accumulation time) increased by more than 30% while a
20-min pre-incubation with MTX resulted in only a 6% increase in intracellular 5FU.
Therefore, MTX is not just a modulator of 5FU anabolism in the tumor but also
shows great potential as an efficient modulator of 5FU transport into tumor cells.
142
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The anabolism of 5FU to its fluoronucleosides was enhanced after pre-incubation of
cells with MTX. Within just the first minute of incubating cells with 1 mM 5FU after
a 20-min pre-incubation with MTX, trace amounts of the fluoronucleosides could
already be detected at a chemical shift of 39.3 ppm, about 3 - 4 ppm downfield from
5FU, phased in the opposite direction (Fig. 4.24).
In conclusion, these studies have demonstrated the presence of two mechanisms for
5FU transport into Walker 256 tumor cells: a saturable carrier-mediated active
transport process and an equilibrative transport mechanism mediated through the ei
nucleoside transporter. The active transport of 5FU into Walker 256 tumor cells
documented in these studies appears to be the general mechanism of 5FU transport
for tumor cells in vitro and may also be the mechanism by which tumors in vivo
transport 5FU.
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CHAPTER VII
7.0 FUTURE DIRECTIONS
With a good understanding of the mechanism by which 5FU is transported into
tumor cells, the next goals would be the search and identification of modulators that
could selectively enhance 5FU transport into the tumor. While these studies have
identified MTX as a potential modulator of 5FU transport, the effect of MTX on the
transport of 5FU needs to be more fully characterized. Questions such as:
(I) Is MTX equally effective in enhancing 5FU transport at all concentrations, (ii)
what is the optimum concentration of MTX required to exert the highest modulating
effect on 5FU transport in to the tumor cells, and (iii) what is the optimum pre
exposure time of cells to MTX to observe the highest enhancement of 5FU transport,
need to be answered.
In addition to MTX as a modulator of 5FU transport, other agents need to be
identified that have the ability to selectively enhance the transport of 5FU into tumor
cells without negatively impacting its chemotherapeutic effectiveness.
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APPENDIX
TABLE OF 1 9 F-CHEMICAL SHIFTS OF FLUORINATED PYRIMIDINES
AND OF OTHER FLUORINATED COMPOUNDS TESTED
Reference Compound 5-Fluorouracil CF3 COOH CFCI3 Molecular
set at 0 . 0 0 set at 0 . 0 0 set at 0 . 0 0 Weight
CF3 COOH 93.9 0 . 0 -75.0 143
4-Fluorobenzophenone 65 -29.9 -103.9 2 0 0
4-Fluorobenzoate 59.1 -34.8 -109.8 140
Fluorobenzene 55.7 -38.2 -113.2 96
Gemcitabine 51.7 -42.2 -117.2 264
F"
50.2 -43.6 -118.7 <19>
5-Fluorotryptophan 40 -53.9 -128.9 2 2 2
1 ,2 -difluorobenzene 29 -64.9 -139.9 114
2-Fluoropyridine 15.9 -78 -156.4 97
Glucuronides of 5FC 7.2 -86.7 -161.7
Hexafluorobenzene 6.9 -87.0 -163.2 186
Glucuronides of 5FU
Nucleotides of 5FU 4.8 -89.1 -164.1 342
(eg FUMP)
5-Fluororotic acid 4.4 -89.5 -164.5 174
Nucleosides of 5FC 4.3 -89.6 -164.6 278
Nucleosides of 5FU 3.7 -90.2 -165.2 263
(eg FUR)
5-Fluoro-2’3’-dideoxy- 3.3 -90.6 -165.6 230
cytidine (FddC)
5-Fluorocytosine (5FC) 1.4 -92.5 -167.5 130
5-Fluorouracil (5FLS) 0.0 -93.9 -168.9 130
3-fluoro-octane - 1 2 . 1 -106.0 -181.0 132
Fluoro-ureido-propionic acid
(FUPA) -17.4 -111.3 -186.3 150
Fluoro-beta-alanine -18.8 - 1 1 2 . 8 -187.7 107
(FBAL)
Fluorocitrate -22.3 -116.2 -191.2 2 1 0
6 -hydroxyfluorocytosine -26.9 - 1 2 0 . 8 -195.8 146
(6 -OHFC)
Dihydrofluorouracii -32.1 -126.2 - 2 0 1 132
(DHFU)
Fluoroacetate -48.2 -142.1 -217.1 78
Downfield: +, left of compound; Upfield, right of compound.
Thus, FNuc is downfield from 5FU, and FBAL is upfield.
References and possible additional sources:
F.A. Bovey, NMR Data Tables for Organic Compounds. 1967
F.A. Bovey, Nuclear Magnetic Resonance Spectroscopy. 1969
W. Bruegel, Nuclear Magnetic Resonance Spectra and Chemical Structure. 1967. p195-210
J.J. Burke and T.R. Krugh: A Table of 1 9 F Chemical Shifts.
158
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Creator Ikonte, Chioma Jane (author)

Core Title Studies on the mechanism and regulation of the transport of 5-fluorouracil (5FU) into tumor cells

Degree Doctor of Philosophy

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