EQUILIBRATIVE-SENSITIVENUCLEOSIDE TRANSPORTER FUNCTION AND REGULATlON IN GEMCITABINE SENSlTlVîTY AND RESISTANCE: IS THERE A POTENTIAL THERAPEUTRC BENEFIT FOR PANCREATIC CANCER? David R. Rauchwerger A thesis submitted in conformity with the requirements for the ûegree of Master of Science, Graduate Department of Pharrnacology, University of Toronto @ Copyright by David R. Rauchwerger (1999)
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EQUILIBRATIVE-SENSITIVE NUCLEOSIDE TRANSPORTER FUNCTION AND REGULATlON IN GEMCITABINE SENSlTlVîTY
AND RESISTANCE:
IS THERE A POTENTIAL THERAPEUTRC BENEFIT FOR PANCREATIC CANCER?
David R. Rauchwerger
A thesis submitted in conformity with the requirements for the ûegree of Master of Science,
Graduate Department of Pharrnacology, University of Toronto
@ Copyright by David R. Rauchwerger (1999)
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Equilibrative-Sensitive Nucleoside Transporter (eeM) Function and Regulation in Gemcitabine Sensitivity and Resistance: b lhere a Potential
Therapeutic Benefit for Pancreatic Cancer? David R. Rauchwerger
ûepartment of Pharmacology University of Toronto
Abstract
Salvage of preformed nucleosides requires transport across the plasma
membrane by specific transport proteins and subsequent conversion to their ribo-
and deoxyribonucleotide foms. In mammalian cells, plasma membrane
transport occurs by sodiumdependent (concentrative) and sodium-independent
(equilibrative) mechanisms. These transport systems are also the route of
cellular uptake for many synthetic nucleoside analogue agents used in cancer
treatment, including gemcitabine (a novel deoxycytidine analogue). This thesis
examines the in vitro effects of gemcitabine on cytotoxicity and the modulation of
these effects by the es-nucleoside transporter and two DNA synthesis inhibitors
(5-fluorouracil and tomudex), with a specific focus on pancreatic cancer.
Cytotoxicity was assessed by clonogenic assay in one human bladder
(MGH-U1 ) and three human pancreatic cancer cell lines (PANC-1, HS-766T, PK-
8). Basal levels of es-NT were quantified in al1 four cell lines by flow cytometric
analysis. Combination experiments were cawied out to determine if upregulating
the es-NT modulates increases in sensitivity to gemcitabine. In two pancreatic
cell lines (PANC-1, HS-766T), treatment with 5-FU followed by gemcitabine
yielded increased cytotoxicity. This effect was also seen in the HS-7661 cell line
when pre-treated with tomudex. For these concentrations of 5-FU and tomudex,
es-NT content was found to be increased over basal levels.
Acknowledgements
I would first like to thank my supervisor, Dr. Malcolm Moore for his constant encouragement. guidance and support during the past two yean. Your comments. criticisms and suggestions were instrumental in my development as an independent, critical, research scientist.
I would also like to thank my advisor. Dr. Gerald Goldenberg and the members of my thesis defense cornmittee, Dr. David Riddick, Dr. David Hedley, Dr. Ted lnaba and Dr. Patricia Harper for their feedback and suggestions.
Special thanks go to Juliet Sheldon for her help with flow cytornetry and Pat for ail her assistance, expertise and. of course, patience through my stay at OCI.
Finally, many thanks go to rny family for their support, love and encouragement that I have always had and will continue ?O enjoy.
. . Abstract ............................................................................................................. II ... Acknowiedgernents ........................................................................................... 111
Table of Contents ............................................................................................. iv List of Tables .................................................................................................... vi List of Figures .................................... ... ........................................................... vii . . ... List of Abbreviations ........................................................................................ vu1
. . 1.4.1 Gemc~ta bine ............................................................................... -7
1 .4.1 -1 Mechanism of Action of Gemcitabine ........................... 7 1.4.1.2 Drug Resistance and Gemcitabine ............................ -13
................................. 1 .4.1.3 Phase I Studies of Gemcitabine 15 ...................... 1 .4.1 -4 Phase II and lil Studies of Gerncitabine 16
3.2.1 Single Drug Exposures ............................................................ -46 3.2.2 Drug Corn bination Exposures .................................................. -47
Table 5.1 6: Human Bladder, Colon and Pancreatic Tumour Cell Line Doubling Times and 5-FU and Tomudex Sensitivities ....... . . . . .. . .. . .. .. .... ........ 85
Append ix:
Table 1 : Staastical Differences Between Basal and Drug-Treated Levels of es Nucleoside Transporter in the MGH-U1 cell line .......... ............. 97
Table 2: Statistical Differences Between Basal and Drug-Treated Levels of es Nucleoside Transporter in the PANC-1 cell line ........................ 97
Table 3: Statistical Differences Between Basal and Dnig-Treated Levels of es Nucleoside Transporter in the HS-766T cell line . . . . . . . . . . . . . . . . . . . . . . -98
Chernical Structures of Deoxycytidine. Ara-C and Gemcitabine .... 8 Metabolic Scheme of Gemcitabine .............................................. 10
............................ Self-Potentiation Mechanisms of Gemcitabine 12 ......................... Chernical Structures of Uracil and 5.Fluorouracil 20
Mechanism of Action of 5-Fluorouracil ......................................... 22 Chernical Structure of Tomudex .................................................. 24 Mechanism of Action of Tomudex ................................................ 26
................ Structures of Nucleosides and Nuclecside Analogues 29 ..................... Chemica f Structure of 5-(SAENTA-x8)-Fluoresœin 36
Growth Curve for MGH-U1 Cell Line .......................................... 5 2 Growth Cuwe for PANGl Cell Line .................................... ... . 5 3 Growth Cuwe for HS-766T Cell Line ................... .. ................... 54
.................................................. Gmwth Cuwe for PK-8 Cell Line 55 Cell Survival Cuwes for MGH-Ul Cell Line ................................ 57 Cell Survival Cuwes for PANGI Cell Line .................................. 58 Celi Suwival Cuntes for HS-766T Cell Line ................................. 59 Cell Survival Cuwes for PK-8 Celi tine ....................................... 60 Combination Studies of Gemcitabine and 5-FU in MGH-U1 ........ 64 Combination Studies of Gerncitabine and Tomudex in MGH-U1 . 65 Combination Studies of Gemcitabine and 5-F U in PANC-1 ......... 66 Combination Studies of Gemcitabine and Tomudex in PANGI .. 67 Combination Studies of Gemcitabine and 5-FU in HS-766T ....... -68 Combination Studies of Gemcitabine and Tornudex in HS-766T . 69 Binding Cuwes of 5x8 in MGH-U1 Cell Line +/- NBMPR ............. 71 Binding Cunres of 5x8 in PANCI Cell Line +/- NBMPR .............. 72 Binding Cuwes of 5x8 in HS-766T Cell Line +/- NBMPR ............. 73 Binding Cuwes of 5x8 in PK-8 Cell Line +/- NBMPR ................... 74 Sample Calibration Curve For RCP-30-5 Rainbow Beads .......... -75
vii
a-MEM 2CdA 5-FU 5x8 ara-C cib
cif
CDP CTP dCK dCMP (d)CMPD dCTP dFdC dFdCDP dFdCMP dFdCTP dFdU dFdUMP DNA dTMP dTrP dUMP ei es es-NT FdUMP FdUTP FITC FPGS FUMP GST HEPES G o G o
a-modification of minimal essential medium 2-chlorodeoxyadenosine 5-Fluorouracil 5-(SAENTA--)-Fluorescein Cytosine arabinoside Concentrative and insensitive to NBMPR, accepts broad array of nucleosides as permeants Concentrative and insensitive to NBMPR, accepts fomycin 6 as a pemeant Concentrative and insensitive to NBMPR, accepts thymidine as a pemeant Concentrative and sensitive to NBMPR Concentrative and sensitive to NBMPR, accepts guanosine as a pemeant Cytidine diphosp hate Cytidine triphosphate Deoxycytidine kinase Deoxycytidine monophosphate (0eoxy)cytidine monophosphate deaminase Deoxycytidine triphosphate 2',2'dlluorodeoxycytidine (gemcitabine) 2'2'-difluorodeoxycytidine diphosphate 2',2'-âifluorodeoxycytidine monophosphate 2',2'difluorodeoxycytidine triphosphate 2',2'difluorodeoxyundine 2',2'difluorodeoxyuridine monophosphate Deoxyribonucleic acid Z'deoxythymidine-5'-monophosphate Deoxythyrnidine triphosphate Z'deoxyuridine-5'-monophosphate Equilibrative and insensitive to NBMPR Equilibrative and sensitive to NBMPR Equilibrative-sensitive nucleoside transporter 5'-fluoro-2'deoxyuridine-5'-monophosphate 5'-f luorodeoxyuridine triphosphate Fluorescein-54sothiocyanate Folyl polyglutamate synthetase Fluorouridine monophosphate Glutathione S-transferase N-(2- hydroxyeth yl(piperazine)-N'-(4-ethanesfonic acid)) Concentration required to inhibit cotony formation by 50% Concentration required to inhibit colony formation by 90%
Leucovorin Multi-drug resistance Molecules equivalent soluble fluorescein MultMrug resistance associated protein S-(p-nitrobenzy1)-64hioinosine; nitrobenzylmercaptopurine n'boside Phosphate-buffered saline P-glycoprotein Reduced folate carrier Ribonucleic acid Ri bonucleotide reductase 5 ' - ~ - ( 2 - a m i n o m e t h y l ) - ~ ~ - ( 4 - n i t r o b e n q l ) ~ i n e Thymidylate synthase Thymidine triphosphate
1.1 Introduction
There exist two pathways of DNA synthesis within human cells: the de
novo and salvage pathways. Salvage of prefomed nucleosides requires their
transport across the plasma membrane and subsequent conversion to their ribo-
and deoxyribonucleotide forms. The efficiency of the salvage pathway of DNA
synthesis can have major effects on cellular sensitivity ta a wide variety of
antirnetabolite drugs because most of these dnigs, including nudeoside
analogues, undergo a generic two-stage metabolism: transpoct into the cell
('"salvage"), followed by phosphorylation to their active triphosphate foms.
Nucieoside analogues taken up by the salvage pathway include cytosine
arabinoside (ara-C), 2-chlorodeoxyadenosine (2CdA) and gemcitabine (dFdC).
Therefore, low activity of this pathway may be a potential mechanism of drug
resistance to these agents. This paper examines the in vitro effects of the novel
nucleoside analogue gemcitabine as a single agent and in combination with two
DNA synthesis inhibitors, 5-FU and tomudex, lod<ing at cytotoxicity and the
effects on the equilibrative-sensitive nucleoside transporter (es-NT). In addition,
schedule dependence of bmg administration was examined in order to provide a
rationale for future clinical trials protocol design.
1.2 Pancreatic Cancer
Adenocarcinorna of the pancreas is the fifth leading cause of cancer-
related deaths in North America exceeded only by lung, colorectal, prostate and
breast cancers (Moore, 1996). Surgery is the only curative treatment currently
available; however, greater than 80% of patients with carcinoma of the exocrine
pancreas are metastatic at diagnosis. Chemotherapy and radiation therapies
rnost commonly play a palliative role in pancreatic cancer care and have not
shown a significant impact on 5-year survival rates (Clark et al., 1996). At
present, pancreatic cancer has the worst 5-year survival rate of any cancer - less
than 5% of al1 pancreatic cancer patients survive five yean (Clark et al., 1996;
Moore 1996).
A variety of drug resistance rnechanisms have been described in
pancreatic tumours. These include P-glycoprotein (P-gp), mufti-drug resistance-
associated protein (MRP), glutaaiione S-transferases (GSTs) and metallothionein
(Collier et el., 1994; Dietel, 1996; Holm et al., 1994; Miller et al., 1 996; Ohshio et
al., 1996; Verovski et al., 1996). P-gp is an integral membrane-bound efflux
pump capable of removing a variety of structurally and functionally unrelated
cytotoxic drugs including the anthracyclines, epidophyllotoxins, alkylating agents
and vinca alkaloids, from tumour cells (Dietef, 1 996; Holm et al., 1 994; Verovski
et al., 1996). Holm et al. (1 994) found that by decreasing the level of mdrl
mRNA expression in a human pancreatic carcinoma cell line resistant to
doxorubicin, one could inhibit the formation of P-gp and reduce the cell'a
resistance to doxorubicin. GSTs function as enzymes of detoxification. By
catalysing the conjugation of potentially mutagenic electrophilic compounds with
reduced glutathione, cells may be protected from toxic cell damage (Dietel,
1996). The Pi class of GSTs (those with acidic isoelectric points) are expressed
by a vanety of human tumours at higher than normal values. It is this isozyme
that is more prominentiy expressed in the majority of pancreatic
adenocarcinornas (Collier et a/., 1994). Because a variety of cytotoxic drugs are
metabolised by GSTs, elevated levels of this enzyme may potentially lead to
tumour dnig resistance.
Over the past ten yean, many new dnigs have been tested for activity
against pancreatic cancer. Over forty phase II studies of new single agents and
combination regimens have been reported (Moore, 1994). None has shown a
response rate greater then 20%, the usual standard that mus! be met for further
testing to be warranted (table 1.2a). In randomized trials, gemcitabine was the
first and only chemotherapeutic agent that has been shown to have any
meaningful impact on either survival or disease related symptoms in pancreatic
adenocarcinorna. Much interest now exists in maximizing the benefit of
gemcitabine against pancreatic cancer and other solid tumours.
(dFdU). (3) Deamination of dFdCMP by dCMP deaminase into 2',2'-
difluorodeoxyuridine monophosphate (dFdUMP). Both dFdUMP and dFdU are
ultirnately broken down into uracil and difluorodeoxyribose (Plunkett et a/., 1995;
Guchelear et al., 1996).
Gerncitabine, abng with its metabolites, interact with a variety of cellular
me!abolic regulatory processes. These interactions serve to enhance the overall
inhibitory actions of gerncitabine on cell viability. Termed 'self-potentiation',
these interactions are not evidenced to such an extent in other anticancer drugs
(Plunkett et al., 1995; Heinemann et al., 1992; Gandhi et al., 1991). The
pathways of gemcitabine self-potentiation and the sites of action of gemcitabine
and its metabolites are summarized in figure 1.4~. The six major mechanisms of
gemcitabine self-potentiation are as follows: (1) dFdCDP is an inhibitory
substrate for ribonucleotide reductase (RR), the enzyme that is responsible for
producing deoxynucleotides required for both DNA synthesis and repair. (2) The
resulting decrease in cellular deoxynucleotides, in particular deoxycytidine
triphosphate (dCTP), is essential in that dFdCTP directly cornpetes with dCTP for
incorporation into DNA by DNA polymerases. This decrease in cellular dCTP,
therefore, increases gemcitabine nucleotide incorporation into DNA. (3) dCK
phosphorylation of gemcitabine is inhibited by deoxycytidine and dCTP
(Heinemann et a!., W88; Bouffard et al., 1993). Therefore, when cellular dCTP
pools are lowered, the rate of gemcitabine phosphorylation is increased resulting
in greater dFdCTP accumulation for incorporation into DNA. These higher levels
of dFdCTP would also act to rnaintain inhibition of ribonucleoside diphosphate
DNA
UTP
--a-- synthetase ,, 1 +
CTP CDP
Gemcitabine dFdU
Figure 1.4~. Self-potentiating mechanisrns of gerncitabine. The numbered pathways are discussed sequentially in the text. The dashed lines indicate inhibitory reactions. RR, ribonucleotide reductase. ---a indicates an inhibitory pathway.
reductase. (4) dCTP is also a required cofactor in the activity of dCMP
deaminase, the rate-limiting enzyme of gemcitabine nucleotide elimination
(Heinemann et al., 1992; Xu et al., 1992). When the cellular levels of dCTP
decline as a result of ribonucleotide reductase inhibition by dFdCDP, dCMP
deaminase activiîy also decreases accordingly. This results in a lower rate of
removal of gemcitabine nucleotides from the celf and most likely contributes to
the retention of the active nucleotides in tumour cells. (5) dFdCTP also functions
to inhibit the dCMP deaminase reaction, adding an additional mechanism that
contributes to the prolonged retention of gemcitabine nucleotides found in tumour
cells (Heinemann et al., 1 992; Gninewald et al., 1 992). (6) dFdCTP inhibits CTP
synthetase at high concentrations, thereby blocking the synthesis of dCTP as
well (Heinemann et al., 1995). This action decreases the available supply of
CDP as a substrate for ribonucleotide reductase, which puts further stress on the
other normal pathways of dCTP formation.
1.4.1.2 Drug Resistance and Gemcitabine
Resistance to gemcitabine may be due to several mechanisms. dCK
deficiency, leading ultimately to decreased dFdCTP formation; increased
elimination of gemcitabine or its monophosphate through increased deamination;
increased dCTP pools, resulting in increased feedback inhibition of dCK; and
finally decreased influx or increased efflux of gemcitabine (Van Haperen et al.,
1995; Van Haperen et al., 1994).
Van Haperen et al. (1995) developed a gemcitabine-resistant human
ovarian cancer cell line in which they found a 10-fou reduction in dCK activity as
compared to the normal, parent cell line. This resistant cell line was found to be
cross-resistant to antimetabolites known to depend on dCK for activation (ara-C,
2-CdA), dFdU (the deamination product of gemcitabine) as well as to cisplatin,
doxorubicin and vincristine (Van Haperen et al., 1995). This may have
implications in future gemcitabine combination Vierapy.
Various lines of evidence have shown an association between increased
cytidine deaminase activity and cellular resistance to cytidine analogues. Firstly,
cytidine deaminase irrevenibly catalyses the deamination of its physiologie
substrates (cytidine and deoxycytidine) as well as nucleoside analogue agents,
including gemcitabine (Camiener, 1967; Chabot et al., 1983; Bouffard et al..
1993). This deamination causes a marked decrease in anti-tumour activity
(Creasey et al., 1966; Muller and Zahn, 1979). Second, in leukaemia patients,
high levels of cytidine deaminase activity have been correlated with resistance to
treatment with ara-C (Steuart and Burke, 1971 ; Onetto et al., 1987). Third,
inhibition of cytidine deaminase in experimental tumours and cefl lines has been
associated with an increased susceptibility to killing by ara-C (Honma et al..
1991 ; Riva et al., 1992). None of these studies, however, has demonstrated a
direct association between cytidine deaminase activity and drug resistance.
Tobias and Blau (1996) performed in vitro studies to determine whether forced
expression of a gene encoding for the enzyme cytidine deaminase can confer
resistance to the cytidine analogues ara4 and gemcitabine. They found that by
overexpressing cytidine deaminase they couM confer at least a 2-fold resistance
to a ra4 and gemcitabine as compared to cells expressing normal levels of this
enzyme.
Sliutz et al. (1996) linked a heat shock protein (hsp70) overexpression
with gemcitabine resistance. Heat shock protein expression is induced in
response to adverse changes in the cellular environment (Lindquist and Craig,
1 988). Hsp70 overexpression has been repoited to induœ cytoprotection in vivo
and in vitro under a wide variety of adverse conditions (Sliutz et al., 1996). In
this study, the cytoprotective effect of hsp70 against gemcitabine was moderate,
with a factor of 2-3 fold. It was shown that by depbting cellular levels of hsp70
using quercetin, a natural flavonoid known to inactivate the heat shock
transcription factor, sençitivity to gemcitabine increased. This increase was
quercetin dosagedependant.
Mackey et al. (1998) showed that nucleoside transporter activity was a
prerequisite for growth inhibition by gemcitabine in vitro. They evidenced two
reasons for this: 1) nucleoside transportdeficient cells were highly resistant to
gemcitabine and 2) treatment of cells that exhibited only equilibrative nucleoside
transporter activity with nitrobenzylthioinosine (NBMPR) or dipyridamole
(equilibrative transport inhibitors, section 2.6) increased gerncitabine resistance.
1.4.1 -3 Phase I Studies (Toxicities)
Various dosing schedules of gemcitabine were evaluated during many
phase I studies. Dose-lirniting toxicities of gerncitabine have k e n found to be
schedule dependent (Eckardt et al., 1995). Poplin et al. (1992) studied two
different therapy regimens: 5-90 mg/m2 twice weekly as a 30-minute infusion and
30-1 50 mg/m2 twice weekly as a bolus injection dunng 5 minutes. The maximum
tolerated doses of gemcitabine for these schedules were 65 and 100 mg/m2
respectively. The dose limiting toxicity was myelosuppression for both schedules
and non-haematological toxicities, including nausea, vomiting and malaise, were
mild (Poplin et al., 1 992).
From other phase I studies, gemcitabine (1 0-1000 mg/m2 as a 30-minute
intravenous infusion weekly for 3 weeks eveiy 4 weeks) was founâ to have
myelosuppression as its major dose-limiting toxicity. Other haematological
toxicities included anaemia and thrombocytopenia. Non-haematological
toxicities found were nausea, vomiting and malaise, which were all mild. The
maximum tolerated dose of gemcitabine was assessed to be 790 mg/m2 for this
dosing schedule (Abbniuese et al., 1991 ; Rosso and Martin, 1994; Pollera et al.,
1994). In studies of previously untreated patients with pancreatic
adenocarcinorna, high-dose gemcitabine (1200, 1500 or 1800 mglm2 given
weekly for 3 weeks every 4 weeks at a constant infusion rate of 10mglm2/min)
elicited fever, myelosuppression, nausea, vomiting and confusion as its dose-
limiting toxicities (Tempo et al., 1994).
1 -4.1 -4 Phase II and III Studies
Phase II studies of the weekly schedule have demonstrated that
gemcitabine has activity against non-small cell lung cancer, bladder cancer,
ovarian cancer, breast cancer and pancreatic cancer (table 1.4a). In pancreatic
cancer, the efficacy of gemcitabine (1000 mglm2 weekly for 7 weeks followed by
1 week of rest and thereafter weekly for 3 weeks every 4 weeks) has been
studied (Rothenberg et al., 1995). 17 of 63 patients (27%) in the study showed a
positive clinical benefit (defined as at least 50% reduction in pain or at least 50%
reduction in daily consumption of analgesics). Clinical benefit, defined as a
composite measure of pain, analgesic consumption, performance status and
weight gain (Hidalgo et al., 1 999). has only recently been incorporated into the
investigation of novel therapies in pancreatic cancer care and recognized as a
valid end point for dnig approval. In a phase III study (Moore, et al., 1995) in
previously untreated patients, gemcitabine was compared to 5-FU. Here, 1 26
patients with advanced pancreatic cancer were randomized to gemcitabine at the
aforementioned schedule or to 5-FU (600 mg/m2 over 30 minutes, weekly). With
gemcitabine, 24% of patients showed a clinical benefit versus 5% of those in the
5-FU a n of the study. Median suwival of the two groups was also reported, and
was found to be 5.65 and 4.41 months respectively, with 18% of gemcitabine
patients alive at 1 year as cornpared to 2% of those who received 5-FU.
1.5 Thymidylate Synthase (TS) Inhibitors
Thyrnidylate synthase (TS) is the rate-limiting enzyme involved in the de
novo synthesis of thymine nucleotides. Inhibition of this enzyme limits the
formation of thymidine triphosphate (TTP), resulting in inhibition of DNA
synthesis. TS has been an attractive target for anticancer drugs since the
Stud y Dooing Scheduk Prior Response Therapy Rate(%)
Non Small-Ce11 Lung Cancer
Anderson et a1 (1 994) 800-1 000 mglm2/wk x 3 wk No 20 (1 6/79)
Abratt et al (1 994) 1000-1 250 mg/m2/wk x 3 wk No 20 (15/76)
Shepherd et al (1 993) 1250 mglm21wk x 3 wk No 20 (1 9/93)
Fosella et a1 (1 993) 1000-1 750 mglm2/wk x 3 wk No 21 (4/19)
Negoro et al (1 994) 1 000-1 250 mg/m2/wk x 3 wk Not stated 30 (1 1/37)
Negoro et al (1 994) 1 000-1 250 mg/m2/wk x 3 wk Not stated 24 (9137)
Lund et al (1 992) 90 mglm2 Nice weekly No 13 (5/40)
Ovarian Cancer
Lund et al (1 994) 800 mg/m2/wk x 3 wk Yes 19 (8142)
Breast Cancer
Carnichael et a1 (1993) 800 mg/m21wk x 3 wk Yes 29 (9135)
Pancreatic Cancer
Casper et ai (1 991 ) 800-1 250 mglm21wk x 3 wk No 13 (5139)
Carnichael et al (1 993) 800-1 000 mg/m2/wk x 3 wk No 9 (2123)
Table i.4a. Phase II trials of gemcitabine.
introduction of the fluoropyrimidines 5-FU and 5-fluorodeoxyuridine by
Heidelberger et al. (Heidelberger et al., 1957). TS inhibition by these compounds
is dependent on their conversion to fluorodeoxyuridine monophosphate (FdUM?)
and the subsequent formation of a temary complex between the enzyme,
FdUMP and the folate cofactor (Langenbach et al., 1972; Danenberg et al.,
1974). In addition to their effects on TS, fluoropyrimidines can also inhibit cell
growth by incorporation into RNA and DNA (Heidelberger et al.. 1957). An
alternative approach to the inhibition of TS has b e n directed toward the
synthesis of analogues of the fobte cofactor. These folate-based TS inhibitors
are specific in their actions and do not possess any of the non-specific actions of
the fluoropyrimidines (Cunningham et al., 1 996; Blackledge, 1 998).
Other factors also favour TS as a chemotherapeutic target. TS levels are
increased in neoplastic cells, implying a greater dependency of tumour cells on
de novo pyrimidine biosynthesis and therefore, enhanced sensitivity to TS
inhibitors relative to normal cells (Hashimoto et al., 1988). In addition, folate-
based inhibiton of TS may also gain selectivity because of the increased
reduced folate transport and polyglutamation capacity in tumour cells (Sirotnak et
al., 1984). These factors should result in increased concentration and retention
of the inhibitors in tumour cells.
1.5.1 5-Fluorouracil
~-FIuo~-c~~..(~-Fu) is a fluonnated pyrimidine antirnetabolite belonging
to the class of anti-metabolites known as TS inhibitors. It was originally
synthesized in 1957 (Heidelberg et ai., 1957). The fluorine substitution occurs at
carbon 5 of the pyrimidine ring in place of a hydrogen (figure 1 5a) .
5-FU is the therapeutic mainstay for colorectal cancer (Schnall and
MacDonald, 1991). It is also used clinically in the treatment of breast, pancreatic
and stomach cancers and squarneous cell carcinoma of the head and neck.
Response rates to 5-FU monotherapy in patients with advanced colorectal
cancer are typically less than 20% (Bleiberg, 1997). Therefore, considerable
interest exists in combining 5-FU with other chemotherapeutic agents in order to
improve therapeutic outcornes. Thus far, efforts to improve efficacy have
included modifying the route or schedule of administration and combining 5-FU
with biochemical modulating agents, such as folinic acid. Gastrointestinal
toxicities (diarrhea, mucositis) are dose-limiting in clinical use.
U raci l 5-FU
Figure 1.5a. Chemical structures of uracil and 5- Ffuorouracil.
1 S.1.1 Mechanism of Action of SIFU
5-FU, like gemcitabine, is an S-phase specific agent. Therefore, dnig
cytotoxicity requires active DNA synthesis and is related to exposure tirne. As 5-
FU is a moâified nucleobase, it must be transported into the cell before it can
exert its cytotoxic actions. This uptake occurs via various nucleoside transport
proteins located at the cell surface (section 1.6; Wang et al., 1997). Once it
enters the cell, three mechanisms exist by which 5-FU exeitç its cytotoxic effects
(figure 1.56): (1) Inhibition of thymidylate synthase (TS) (Heidelberg et aL,
1960a. 19606). This is the primary mechanism of action of 5-FU. Thymidylate
synthase catalyzes the methylation of Tdeoxyuridine-5'-monophosphate
(dUMP) to 2'4eoxythymidine-5'-monophosphate (dTMP) in a reaction which
uses the reduced folate 5,1 O-methylene tetrahydrofolate as a cofactor. 5-FU is
converted intracellularly to 5luoro-2'-deoxyuridine-5'-monophosphate (FdUMP).
FdUMP fonns a covalent temary bond with thymidylate synthase and its reduced
cofactor ~~*'~-methylene tetrahydrofolate. Binding results in enzyme inhibition
and subsequent depletion of deoxythymidine triphosphate (dTTP), thereby
preventing DNA synthesis and celf growth. (2) 5-FU or FdUMP may be
converted to fluorouridine monophosphate (FUMP) which is further
phosphorylated to the triphosphate, FUTP. FUTP may be incorporated into RNA
producing non-functional RNA aiid defects in protein synthesis. (3) FdUMP may
be phosphorylated to the 5'-fluorodeoxyuridine triphosphate (FdUTP) and
incorporated directly into DNA, altering DNA stability (Cheng and Nakayama,
Figure 1.5h Mechanism of action of 5-FU. UK, uridine kinase; UMK, UMP kinase; UDK, UDP kinase; TK, thymine kinase. Numbered pathways correspond to those discussed in the text.
1 983). The DNA repair enzyme uracil DNA glycosylase functions to remove
uracil from DNA and also excises FdUTP resulting in DNA single strand breaks
and DNA fragmentation (Lonn and Lonn, 1 986).
It has been speculated that many of the toxic effects of 5-FU (and its
modulators) are due to the lack of specificity in their actions on thymidylate
synthase (Mead, 1996) and thus a search for more specific TS inhibitors has
been undertaken.
1.5.1.2 Drug Resistance and ÇFU
5-FU resistance has been primarily associated with insufficient inhibition
of thymidylate synthase (Peters et al., 1 995). In vitro and in vivo 5-FU resistance
has also been associated with the following: (1) Decreased accumulation of
FdUMP due to decreased 5-FU activation or increased 5-FU inactivation
(Mul kins and Heidelberger, 1 982). (2) lncreased activity of thyrnidylate synthase
(Chu et aL, 1993; Johnston et al., 1995) and amplification of the thymidylate
synthase gene (Clark et al., 1987; Berger et al., 1985). (3) Changes in
nucleotide pools (Aronow et al., 1984; Kaufmann et al., 1984). (4) Point
mutations in the gene encoding for thymidylate synthase, resulting in an enzyme
with an altered structural fom and lower affinity for both FdUMP and the cofactor
~~*'~-rnethylene tetrahydrofolate. Binding of 5-FU in the absence of this cofactor
results in an unstable binary cornplex. FdUMP then becomes a weak inhibitor of
thymidylate synthase.
1.5.2 Tomudex (Raltbeulcl)
The biosynthesis of thymidine monophosphate (TM P) requires 5,100
methylene tetrahydrofolate, which serves as a cofactor in the TS-catalysed
transfer of a one-carbon unit to dUMP. Due to the Iimited success of
fluoropyrimidine substrate analogues (such as 5-FU) in the treatment of
colorectal and other cancers (Rusturn and Creaven, 1988), analogues of the
folate cofactor of TS were developed. Non-specifk, non-TS effects of 5-FU (and
its modulators) on RNA are believeâ, as previously noted. to account foi some
aspects of toxicity seen during therapy with these antimetabolites, such as
mucositis and rash. A specific TS inhibitor, which does not require modulation
and does not possess non-specific actions on RNA, presents an attractive
research goal. Tomudex, a quinazoline folate analogue (figure 1.5~). is a new
chemotherapeutic agent that aims to rneet this research target. It entered phase
I evaluation in Europe in 1991 (Jackman et al., 1995). Tomudex is a folate-
based TS inhibitor and is an analogue of the folate cofactor for TS. It is a highly
specific, laboratorydesigned TS inhibitor that does not require modulation and
which does not have non-specific effects on RNA (Cunningham et a/., 1996;
Blackledge, 1 998).
Figure 1 .Sc. Chemical structure of tornudex.
24
Tomudex has been approved for the treatment of advanced colorectal
cancer and is currently k i n g studied in a wide vanety of malignancies including
squameous cell head and neck carcinoma, prostate and gastric cancers and
pancreatic cancer. In patients with advanced colorectal cancer, Tomudex has a
response rate similar to 5-FU (Blieberg, 1997; Cunningham et al., 1996) when
used as a single agent. In pancreatic cancer, studies have shown that Tomudex
has a response rate of 12% (Cunningham et al.. 1996). As with 5-FU. there is
considerable interest in combining Tomudex with other chemotherapeutic agents
in an attempt to improve therapeutic outcomes. Adverse effects of Tomudex
include fatigue, leukopenia, thrombocytopenia and diarrhea.
1 S.2.1 Mechanism of Action of Tomudex
Tomudex is a mixed noncornpetitive inhibitor of TS (Duch et al., 1993).
The mode of action of tomudex is specific and is based on cornpetition with 5 1 0-
methylene tetrahydrofolate for TS. Tomudex is transported into cells via a
reduced folate carrier (RFC) and is rapidly and extensively polyglutamated by the
The effect of combining gemcitabine with either 5-FU or tomudex at a
range of concentrations was detemined by exposing cells to the combination
either concurrently for 24 hours, or sequentially, with each exposure lasting 24
hours. Table 4.3a illustrates the cytotoxicity of the agents used when acting
alone (data estimated from log concentration-responçe curves, figures 4.2a-c).
Concentration [pM]
Figure 4.28. Log concentration-response curves following 24 hour exposure of the MGH-Ul cell line to (i) gemcitabine. (a) 5-FU and ( A ) tomudex. Cytotoxicity was detennined by the colony-forming assay. Each point represents the rnean of three independent experiments; each line has an ? value 2 0.90.
Concentration [pM]
Figure 4.26. Log concentration-response curves following 24 hour exposure of the PANC-1 cell line to (m) gemcitabine, (e) 5-FU and ( A ) tomudex. Cytotoxicity was determined by the colony-fonning assay. Each point represents the mean of three independent experiments; each line has an 8 value 2 0.90.
Concentration pM
Figure 4 .2~ . Log concentration-response curves following 24 hour exposure of the HS-766T cell line to (a) gemcitabine, (a) 5-FU and (A) tomudex. Cytotoxicity was detennined by the colony-fonning assay. Each point represents the mean of three independent experiments; each line has an P va!ue 2 0.90.
Concentration [pJM]
Figure 4.2d. Log concentration-response curves following 24 hour exposure of the PK-8 cell line to (m) gemcitabine, (a) 5-FU and (A) tomudex. Cytotoxicity was deterrnined by the colony-foming assay. Each point represents the mean of three independent experiments; each line has an ? value 2 0.90.
Table 4.2a Dnig concentrations required to inhibit colony formation by 50% following 24 hour dnig exposures. Values were estimated from the log concentration-response cuwes.
Table 4.2b. Dnig concentrations required to inhibit colony formation by 90% following 24 hour dnrg exposures. Values were estimated from the log concentration-response curves. - indicates ICgO concentration was not achieved.
Table 4.3a. Concentration of agents used in combination expenments. Numbers in parentheses indicate cell survival when used as a sing!e agent (estimated from log concentration-response CU ives in figures 4.2a-c).
As shown in figures 4.3af pre-treatment of the HS-766T cell line with
either 5-FU or tomudex augmented the effects of sole gemcitabine treatment
along with the PANC-1 cell line when pre-treated with 5-FU. The concurrent and
gemcitabine pre-treatment regimens showed no added benefit over single agent
gemcitabine treatrnent in these cell lines. In the MGH-Ul cell line, no
augmentation in cytotoxicity was seen when 5-FU or tornudex was combined in
any manner with gemcitabine.
Figure 4.38. Combination studies of gemcitabine (0.8 pM) and 5-FU in the MGH-U1 cell line following both concurrent (24 hour) and sequential (24 hours per exposure) exposures. Results are expressed as % colony foning efficiency and are the mean f S.O. of three or more independent experiments.
100 Gemcitabine 4 Tomude
100 Gemcitabine + Tomude
.I .
.I .
Figure 4.36. Combination studies of gerncitabine (0.8 pM) and tomudex in the MGH-U1 cell line foilowing both concurrent (24 hour) and sequential (24 hours per exposure) exposures. Results are expressed as % coiony forrning efficiency and are the mean f S.D. of three or more independent experiments.
Gemdtaôine + 5Çl.i -r- -
Figure 4.3~. Combination studies of gemcitabine (1 phd) and 5-FU in the PANC-1 cell line following boai concurrent (24 hour) and sequential (24 hours per exposure) exposures. Results are expressed as % colony foning efficiency and are the mean f S.D. of three or more independent experiments.
Gemdtabine Tomudex
T T - T
- -
1 Lt
10 - 4
100 1OOO
Gemcitabine + Tomudex t
Figure 4.3d. Combination studies of gemcitabine (1 pM) and tomudex in the PANC-1 cell line following both concurrent (24 hour) and sequential (24 hours per exposure) exposures. Results are expressed as % colony forming efficiency and are the mean f S.D. of three or more independent experiments.
Figure 4.3e. Combination studies of gemcitabine (0.4 pM) and 5-FU in the HS- 766T cell line following both concurrent (24 hour) and sequential (24 hours per exposure) exposures. Results are expressed as % colony forming efficiency and are the mean t S.D. of three or more independent experiments.
100 Gemcitabine Tomude
100 Gerncitabine + Tomudex
a
I
100 Tomudex a Gemcitabi
a a
10 b 7- -, - - - - . - - - - -
9
1 7
- . . . a . - . . . . , . . . . . . .
9 '. * . . . . . . . . . . - ; - ; < a . *
O- 1 . 8
- 1 . 1 8
O 1 10 100 1000
Figure 4.3t Combination studies of gemcitabine (0.4 pM) and tomudex in the HS-766T cell Iine following both concurrent (24 hour) and sequential (24 houn per exposure) exposures. Results are expressed as % colony foming efficiency and are the mean & S.D. of three or more independent experiments.
Figure 4.4s Binding cuwes of 5x8 in the (i) absence and (a) presence of NBMPR in the MGH-U1 cell fine. Specific binding may be calculated by subtracting non-specific binding (in the presence of NBMPR) from total binding (in the absence of NBMPR).
O 5 10 7 5
[SAENTA] (n M)
Figure 4.46. Binding curves of 5x8 in the (i) absence and (a) presence of NBMPR in the PANC-1 cell line. Specific binding may be calculated by subtracting non-specific binding (in the presence of NBMPR) from total binding (in the absence of NBMPR).
O 5 10 15 20
[SAENTA] nM
Figure 4.4~. Binding curves of 5x8 in the (i) absence and (4 presence of NBMPR in the HS-766T cell line. Specific binding may be calculated by subtracting non-specific binding (in the presence of NBMPR) from total binding (in the absence of NBMPR).
[SAENTA] nM
Figure 4.4d. Binding curves of 5x8 in the (i) absence and (a) presence of NBMPR in the PK-8 cell line. Specific binding may be calculated by subtracting non-specific binding (in the presence of NBMPR) from total binding (in the absence of NBMPR).
MESF
Figure 4Ae. Sample calibration cuwe for flow cytornetric analysis using the rainbow calibration beads (RCP-30-5). 6 > 0.99.
Table 4.48. Specific binding of SAENTA (molecules equivalent soluble fluorescein (MESF) per cell) and gemcitabine sensitivities for four human cancer cell lines prior to treatment with dnig. These correspond to basal levels of cell surface es-NT and lCso values respectively.
Following treatment of the MGH-UI, PANC-1 and HS-766T cell lines with
varying concentrations of either gemcitabine, tornudex or 5-FU. cells were
analyzed for es-NT content. As shown in table 4.46, treatment of the PANC-1
cell line with 1 pM dMC and 30 pkî 5-FU for 24 houn showed a significant (p c
0.05) increase in cell-surface es-NT content over basal levels. In addition,
treatment of the HS-766T pancreatic carcinoma cell line with 30 pM and 100 pM
5-FU and 100 nM and IWO nM tomudex afl resuited in a significant (p < 0.05)
increase in es-NT sites versus control levels. AH other treatment groups for
these three cell lines showed no difference in es-NT content compared with
basal levels.
T m n t Factor Incmu6 In iT MESF ww oontrd-
20 5-FU 0.93 20 jN5-FU x 48 hr 0.70
40 PM 5-FU 1 .IO 1 O0 5-FU 1.57
100 nM Tomudex 1 -66 1 pM Tomudex 1.41
10 nM Gemcitabine 0.96 800 nM Gemcitabine 1 .O3
30 5-FU 1 O 0 W5-FU
100 nM Tomudex 1 pM Tomudex
40 nM Gemcitabine 1 pM Gemcitabine
30 ~J.M 5-FU 1 O 0 @l5-FU
100 nM Tomudex 1 phi Tomudex
Table 4.4b. Factor increase over control in specific binding of SAENTA after treatment with either 5-FU, tomudex or gemcitabine. All incubations were for 24 hours unless otherwise indicated. ** denotes statistically significant difference (p c 0.05, t-test) behnreen treatment group and control.
Characteflzing the various factors that affect cellular differences in
cytotoxicity to a chemotherapeufc agent can provide insight into how one might
modulate these effects. This thesis focused on the nucleoside transporter.
Specifically. the role that the es-NT plays in gemcitabine cytotoxicity. We
hypothesized that the sensitivity to gemcitabine would be influenced by basal
levels of es-NT. If this proved tnie, it would follow that manipulation of the
amount of es-NT couM alter cellular sensitivity to gemcitabine.
DNA synthesis inhibitors such as 5-FU and tornudex have been previously
shown to upregulate the number of cell surface es-NTs (Pressacco et a/..
1995a). These results point towards the usefulness of these agents as possible
modulators of gemcitabine tuxicity. We hypothesized that if we could increase
the amount of es-NT on the cell surface, then greater amounts of gemcitabine
would enter the cell leading to increased cytotoxicity. In examining this
hypothesis, it is important to realize that both 5-FU and tomudex are cytotoxic
antirnetabolite drugs that will exert their own cytotoxic actions on de novo DNA
synthesis in addition to any nucleoside transporter-related effects. These
cytotoxic effects m u r through different mechanisms than those caused by
gemcitabine; TS inhibition versus incorporation into DNA. We therefore are
interested in these drugs' effects on the es-NT and in their combined cytotoxic
effects so that we may gain additional insight into this novel combination
regimen.
5.1 Single Drug Cytotoxicity
The cytotoxicity of gemcitabine, 5-FU and tomudex in the MGH-U1,
PANC-1, HS-766T and PK-8 cell lines was initially detemined. Growoi inhibition
was found to be concentration dependent for all three drugs in al1 four cell lines.
The log concentration-response curves for tomudex in the MGH-U1, PANC-1
and HS-766T cell lines as well as that for 5-FU in the HS-766T cell line appeared
to reach a plateau. In addition, the log concentration-response curves for al1
three drugs in the PANC-1 cell line seemed to reach a plateau. The log
concentration-response curves for gerncitabine and 5-FU continued to decline for
al1 other cell lines, an indication that maximal cytotoxicity has not yet been
attained. Due to this plateau effect mentionad earlier, lCw values were not
obtained for al1 drugs in every cell line.
All the drugs used in this study belong to the anti-metabolite class of
oncolytic agents. These agents ail act as inhibitors of macromolecule
biosynthesis which block cellular replication. Antimetabolites generally compete
with endogenous substrates in cellular metabolic processes to accomplish this
goal. Gemcitabine is in cornpetition with dCyd, 5-FU with dUMP and tomudex
with the endogenous cofactor for TS (5,lO-methylene tetrahydrofolate). For the
TS inhibitors 5-FU and tomudex, it is not surprising to observe a plateau in the
log concentration-response curves. Once TS is completely inhibited, cytotoxic
effects are at a maximum and additional drug present in the cell will not
significantly enhance these effects. Gemcitabine must first be taken up into the
cell via a saturable transport process. Once transport has reached saturating
levels, any additional dnig present will not serve to increase cytotoxicity.
Therefore, we would again expect a plateau to occur in the log concentration-
response plots for gemcitabine. In those cell lines having greater amounts of
functional transporters we would expect this plateau effect to occur farther along
the log concentration-response curve. This trend, however, was not observed
and could be accounted for by non-functional transporter present in the cells or
by decreased dCK eff iciency .
5.1 -1 Gemcitabine
The log concentration-response cuntes for gemcitabine are shown in
figures 4.2ad. The MGH-U1 cell line was the most sensitive to gemcitabine at
its lCso concentration (1.5 nM), having an IC50 value 2.7, 26.7 and 2.3 x 1 o4 times
lower than for the HS-766T, PANC-1 and PK-8 cell lines respectively. No lCso
value was reached for the PK-8 cell line. For the other cell lines, the relative
sensitivities to gemcitabine changed so that an lCgo value of 250 nM for the HS-
766T cell line was 3 and 16 fold lower than those for the MGH-U1 and PANC-1
cell lines respectively (tables 4.2a,b).
Gemcitabine has also been tested in several other human cell lines
including ovarian (A2780, OVCAR-3), colon (WiDr, C26-10) and squarnous cell
gemcitabine sensitivities (24 hour exposure) in these cell lines with those in our
cell Iines at the ICW level. Aside from Our gemcitabine resistant PK-8 cell line,
the IC5* values al1 fall in the nanomolar range.
. :-aiII Une
Table 5.18. Cell doubling times and sensitivities to gemcitabine after 24 hour exposure in several human tumour cell lines. Cell lines defined in text. Data for cell lines not from this work taken from Ruiz Van Haperen et al., 1994.
Gemcitabine is a cell cycle specific agent, with maximal cytotoxicity
occurring during S-phase. We would therefore expect cell lines with shorter
doubling times to be more sensitive to the cytotoxic actions of gemcitabine. The
MGH-U1 cell line has the shortest doubling time, followed by the PK8, PANC-1
and HS-766T cell lines. This, however, does not correspond with cell
sensitivities
at the lCsd values. This agrees with previous data from other human carcinoma
cell Iines mentioned eariier which also shows no correlation between cell
doubling time and gemcitabine sensitivity (Ruiz Van Haperen et ai.. 1994) (Table
5.1 a).
5.1.2 SIFluorouracil
With the exception of the PANC-1 cell Une, the log concentration-response
curves of 5-FU continue to decrease at the highest drug concentrations used,
indicating that maximal cell cytotoxicity has not yet b e n reached (figures 4.2a-
d). 5-FU was most sensitive at its ICx, value of 230 nM in the PANC-1 cell line.
This was 2.2, 12 and 17.4 fold lower than the lCso values in the MGH-U1, HS-
766T and PK-8 cell lines respectively. An ICgO value was only reached in the HS-
766T and PK-8 cell lines and was 55 pM and 65 pM respectively (table 4.2ab).
5-FU has b e n well studied in human colon tumour cells. A cornparison of
our bladder and pancreatic cell line sensitivities with several colon carcinoma cet1
lines may be seen in table 5.1 b. The pancreatic cell lines used for this work are
more sensitive to 5-FU than almost al1 of these colon tumour cell lines.
Table 5.1 b. Cell doubling times and sensitivities to 5-FU and tomudex following 24 hour exposure in several human tumour cell lines. Cell fines defined in text. Data for cell lines not from this work taken from Van Triest et al., 1999.
5-FU is also a cell cycle, S-phase specific cytotoxic agent and like
gemcitabine, dnig sensitivity in the four cell lines used and those listed in table
5.1 b could not be explaineâ by doubling times.
5.1.3 Tomudex
The log concentration-response curves for tomudex reached a plateau in
ail cell lines except for PK-8, in which maximal cytotoxicity was never attained
(figures 4.2a-d). At its lCso concentration, tomudex was most sensitive in the
PANC-1 cell line (0.02 nM), which had an lCso value 2, 150 and 1OOO fou lower
than for the PK-8, HS-766T and MGH-U1 cell lines respectively. Only in the PK-
8 cell line was an IC90 value reached (150 nM) (tables 4.2a.b).
Like 5-FU, tomudex has been studied extensively in human colon cancer
cells. Table 5.1 b shows that al1 of the pancreatic cancer cell lines used in our
study were more sensitive to tomudex than the colon carcinoma cells. This
indicates that tomudex may prove to be a beneficial agent in chemotherapy of
the pancreas. Once again, cell cytotoxicity for a cell cycle specific agent did not
correlate to cell doubling times.
5.2 Drug Combinations
In designing a clinically useful combination therapy regimen, it is desirable
that the dmgs have different, non-overlapping toxicities and different
mechanisms of action. Having non-overiapping toxicities will ideally allow us to
use the maximum tolerated dose of each individual drug when designing the
combination dosing schedule. If the drugs had similar toxicities, we would have
to roll back the dosage of each drug when combining them in order to make the
regimen tolerable to the patient. Possessing different mechanisms and targets of
action prolongs the development of resistance to the therapy because there exist
two separate rnechanisms and cellular processes to which resistance must
develop against For the combination regimens studied in this work (5-FU &
gemcitabine; tomudex & gemcitabine) the dmgs involved possessed non-
overfapping toxicities and differing mechanisms of action. This is one reason
that 5-FU and tomudex make good candidates for combination therapy with
gemcitabine.
In order to establish a combination regimen showing increased efficacy
against pancreatic tumours over gemcitabine alone, gemcitabine was combined
with either 5-FU or tornudex bath concurrently and sequentially in three human
tumour cell lines (MGH-U 1, PANC-1 and HS-766T). Ceil cytotoxicity following
these combination exposures was dependent upon the cell line, single agent
concentrations used and sequence of drug administration. For al1 combination
experiments, a constant concentration of gemcitabine was used within each cell
line while varying the concentration of the DNA synthesis inhibitor (5-FU or
tomudex) (Table 4.3a). This was done to detemine at what concentration(s), if
any, would 5-FU or tomudex increase cellular sensitivity to gemcitabine.
Pressacco et al. (1 9956) have previously reported that DNA synthesis inhibitors,
including 5-FU and tomudex, will upregulate es-NT expression in the MGH-U1
cell line. Based on this observation, we hypothesized that if we could upregulate
the es-NT'S by pre-treatment with either 5-FU or tomudex, we could increase
gemcitabine cytotoxicity.
5.2.1 Gemcitabine and 5-FU
A single concentration of gemcitabine (ICao, ICW, lCse concentration in the
PANC-1, MGH-U1 and HS-766T cell lines respectively) was combined with a
range of 5-FU concentrations from 0-100 pM (Table 4.3a). In ths MGH-U1 cell
line, no increased effects were seen during any schedule of treatment with 5-FU
and gemcitabine, as the cell survival remained approximately the same as for
gemcitabine treatment alone (10%). At higher concentrations of 5-FU in the
PANC-1 and HS-766T cell lines (30, 100 pM), there appeared to ôe an
interaction between the two drugs when they were administered in sequence,
where 5-FU was given first (figures 4.3a,cte).
5.2.2 Gemcitabine and Tomudex
A single concentration of gemcitabine (as above) was combined with
tomudex concentrations ranging from 0-1000 nM. In the MGH-U1 and PANC-1
cell lines, no increased effects were seen versus single agent gemcitabine
treatment with any of the schedules. At the upper range of concentrations of
tomudex in the HS-766T cell line (0.1, 1 PM), increased cell kill was observed
over gemcitabine alone when the cells were pre-treated with tomudex (figures
Table 1: Is there a statistically significant increase in the number of cell surface es nucleoside transporters after treatment as compared to basal levels in the MGH-U1 cell line. Bolded figures indicate statistically significant differences.
Treatment P.Valus
100 pM 5-FU 0.0562
1WnMTomudex 0.1027 '
1 pkl Tomudex 0.0867
40 nM dFdC 0.0561
1 pM dFdC 0.01 68
Table 2: Is there a statistically significant increase in the number of cell surface es nucleoside transporters after treatment as compared to basal levels in the PANC-1 cell line. Bolded figures indicate statistically significant differences.
1 Treatment P-V-ùe 1 30 pM )FU
100 5-FU O.ml7 100 nM Tornudex
Table 3: Is there a statistically significant increase in the nurnber of cell surface es nucleoside transpoiters after treatment as compared to basal levels in the HS- 766T cell Iine. Boldad figures indicate statistically significant differences.
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