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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2004 Jun 15;130(8):453–459. doi: 10.1007/s00432-004-0575-6

Effects of oxaliplatin and CPT-11 on cytotoxicity and nucleic acid incorporation of the fluoropyrimidines

Manish Patel 1, Ram Agarwal 1, Bach Ardalan 1,✉
PMCID: PMC12161872  PMID: 15205948

Abstract

Purpose

The addition of oxaliplatin or CPT-11 to 5-FU has become common practice in the treatment of colorectal cancer. It is not known, however, which fluoropyrimidine drug (5-FU, FUdR, or FUR) will produce superior cytotoxicity when combined with either oxaliplatin or CPT-11. The purpose of the study was to determine the effects of oxaliplatin and CPT-11 on cytotoxicity and nucleic acid incorporation of all three fluoropyrimidines.

Methods

HT-29 cells were exposed for 2 h to IC10, IC30, and IC70 of oxaliplatin and CPT-11. Subsequently, cells were exposed for 24 h to IC10, IC30, and IC70 of 5-FU, FUdR, and FUR. Cytotoxicity was measured by the MTT assay. Nucleic acid incorporation of [3H]fluoropyrimidine was then compared in the presence and absence of oxaliplatin or CPT-11 pretreatment.

Results

Synergistic cytotoxicity was displayed when IC30 of oxaliplatin or CPT-11 was combined with IC10 and IC30 of the fluoropyrimidines. One fluoropyrimidine did not achieve superior cytotoxicity over the others. After pretreatment with oxaliplatin or CPT-11, cytotoxic antagonism was observed as the concentration of a fluoropyrimidine increased up to IC70. The increasing cytotoxic antagonism correlated with decreases in fluoropyrimidine nucleic acid incorporation. The most significant incorporation difference existed within the 5-FU treated group.

Conclusions

No single fluoropyrimidine is more cytotoxically effective over the others when combined with oxaliplatin or CPT-11. Correlation of cytotoxic antagonism to the inhibition of fluoropyrimidine nucleic acid incorporation implies difficulties in drug transport and/or metabolism only after oxaliplatin or CPT-11 pretreatment.

Keywords: Oxaliplatin, CPT-11, 5-FU, FUdR, FUR

Introduction

Colorectal cancer is one of the leading causes of cancer death in the world. Palliative chemotherapy remains the only option for the majority of patients. For the last 40 years, 5-Fluorouracil (5-FU) has been the most commonly used chemotherapeutic agent in the treatment of colorectal cancer (Schmoll et al. 1999). However, the response rate to this drug has usually been under 25% and patient survival has not been significantly prolonged (Link et al. 1988). The inadequate clinical results observed with 5-FU could be attributed to its relatively slow anabolism to the active metabolites in most cells. Clinical dissatisfaction with the drug also stems from increased cases of resistance. Figure 1 illustrates the pathways through which the fluoropyrimidines are activated and incorporated into DNA and RNA. One pathway leads to the conversion to FUdR (5-fluoro-2’-deoxyuridine), which subsequently forms FdUMP. FdUMP is a potent inhibitor of the enzyme thymidylate synthase (TS). This step is essential for DNA synthesis and its inhibition is highly regarded to be responsible for the cytotoxic effects of 5-FU (Langenbach et al. 1972; Pinedo and Peters 1988; Peters et al. 1991; Shuey et al. 1995; Sanguedolce et al. 1998; Sun et al. 2002). The incorporation of 5-FU into RNA represents an alternative pathway in its metabolism. This route has also been accepted as the main explanation for 5-FU’s actions (Houghton et al. 1979; Mandel et al. 1979; Kufe and Major 1981; Glazer and Lloyd 1982; Dolnick and Pink 1983; Geoffroy et al. 1994).

Fig. 1.

Fig. 1

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Metabolic pathways for the fluoropyrimidines. FU, FUdR, and FUR are 5-fluorouracil, 5-fluoro-2’-deoxyuridine, and 5-fluorouridine, respectively. FdUMP is 5-fluoro-2’deoxyuridine-5’-monophosphate and FdUTP is 5-fluoro-2’deoxyuridine-5’-triphosphate. FUMP, FUDP, and FUTP are 5-fluorouridine-5’mono-, di-, and triphosphate, respectively. The enzyme thymidylate synthase is indicated by T.S. The dotted arrow displays the inhibitory action of FdUMP on T.S. activity

As a result of the clinically unsatisfactory effects of 5-FU, a significant amount of interest exists to utilize FUdR or FUR (5-Fluorouridine) as substitutes. FUdR has shown significantly superior antitumor activity in animal models compared with 5-FU (van Laar et al. 1993; van Laar et al. 1998). Arisawa et al. demonstrated higher in vitro activity of FUdR than 5-FU and reduced acquired resistance when treated for a shorter period with combination therapy (Arisawa et al. 1994). In addition, many clinical trials have shown significantly superior response rates for FUdR when compared to 5-FU (Ansfield and Curreri 1963; Buroker et al. 1976; Levin and Gordon 1993). Despite this existing evidence for the potential superiority of FUdR, 5-FU is still mainly preferred due to its assumed lower toxicity and cost. While 5-FU and FUdR are currently used in the treatment of gastrointestinal tumors, the use of FUR has been limited due to its systemic toxicity. However, Link et al. demonstrated cytotoxicity could be achieved with FUR at substantially lower doses and shorter exposure times than with 5-FU (Link et al. 1988). FUR has irreversible and non-phase-specific RNA-directed cytotoxic effects and has displayed more rapid cellular uptake and efficient conversion to nucleotides when compared to 5-FU (Wilkinson and Pitot 1973). In another study, FUR was also found to be more effective at inhibiting DNA synthesis than 5-FU (Kessel et al. 1971). The differences in metabolism, favorable pharmacokinetics, and mechanism of action of FUR have increased its use in treating superficial bladder cancers (Richie 1992; Boring et al. 1994; Song et al. 1997). As with 5-FU and FUdR, the development of resistance to FUR often occurs.

In an effort to improve response rates and survival, there has been an increased effort to find agents to potentiate the effects of the fluoropyrimidines or treat tumors that are unresponsive to the fluoropyrimidines. Oxaliplatin was recently developed for clinical use and is rapidly being utilized for the treatment of colorectal cancer. De Gramont et al. recently established the clinical superiority of a combination of oxaliplatin/5-FU/leucovorin over 5-FU/leucovorin in terms of response rate and progression-free survival (De Gramont et al. 2000). The tested drug sequence for this Phase III clinical trial was oxaliplatin/leucovorin for 2 h followed by 5-FU during 48 h, repeated every 2 weeks. Other studies have also shown superior response rates when oxaliplatin and 5-FU were combined (Levi et al. 1992; Levi et al. 1994; Levi et al. 1997; Levi et al. 1999; Giacchetti et al. 2000). Another new drug that has also recently stimulated interest is irinotecan or CPT-11. Similar to oxaliplatin, CPT-11 alone is active against colorectal cancer and in patients resistant to 5-FU (Cunningham et al. 1998; Rougier et al. 1998). It was recently shown that the combination of CPT-11/5-FU/leucovorin produced a higher response rate (Douillard et al. 2000; Saltz et al. 2000) and prolonged survival (Douillard et al. 2000) than 5-FU/leucovorin in advanced colorectal cancer patients.

Since cytotoxic and clinical success is demonstrated when 5-FU is combined with oxaliplatin and CPT-11, efforts now need to be made to explore the effects of combining FUdR or FUR with oxaliplatin and CPT-11. Researchers have often observed the potential superiority of FUdR and FUR over 5-FU. If oxaliplatin and CPT-11 act as synergistic agents with 5-FU, perhaps similar or superior results will ensue when these drugs are combined with FUdR or FUR. The goal of this study was to determine which fluoropyrimidine acts most synergistically with oxaliplatin and CPT-11. It was expected that it would be possible to establish the concentrations of each drug in a combination that displays superior cytotoxic synergy or antagonism. The MTT cytotoxic assay was utilized with HT-29 colon cancer cells to determine cytotoxicity. Because of the growing evidence for superior clinical success, it was hypothesized that the combination of oxaliplatin or CPT-11 with FUdR would display higher cytotoxic synergy than when the two drugs were combined with 5-FU or FUR. The final aim of the study was to correlate cytotoxic synergy and antagonism with fluoropyrimidine nucleic acid incorporation. After pretreatment with oxaliplatin or CPT-11, nucleic acid incorporation of varying concentrations of [3H]5-FU, FUdR, and FUR was compared. The final nucleic acid incorporation potentially represents each fluoropyrimidine’s ability to be transported and/or metabolized.

Materials and methods

Materials

HT-29 human colon adenocarcinoma cells were purchased from ATCC. RPMI 1640 culture medium was obtained from Gibco. LKT laboratories supplied oxaliplatin and CPT-11. [3H]5-FU, [3H]FUdR, and [3H]FUR were purchased from Moravek Biochemicals. All other chemicals were obtained from Sigma Chemical.

Cell culture

HT-29 human colon adenocarcinoma cells were grown in monolayer cultures in RPMI 1640 media supplemented with 5% heat inactivated Fetal Bovine Serum. No antibiotics were added to the medium. The cultures were incubated at 37 °C in a humidified 5% CO2 atmosphere. The cells were trypsinized and passed once a week. Doubling time was noted to be approximately 24 h.

Individual drug cytotoxicity assay

Prior to determining cytotoxicity for combinations of oxaliplatin or CPT-11 with the fluoropyrimidine drugs, a percentage-inhibition curve and inhibitory concentrations for each of the individual drugs needed to be established. Growth inhibition was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based cytotoxicity assay (Freshney 2000). Briefly, 200 μl of the cell suspension (5×103 HT-29 cells) and 200 μl of RPMI 1640 growth medium were added to each well of a 96-well flat bottom microtitration plate. All plates were incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere. Eight dilutions of each drug (oxaliplatin, CPT-11, 5-FU, FUdR, and FUR) were prepared in growth medium to give eight different concentrations for each drug. Each drug solution was filter sterilized. After incubation, 400 μl of growth medium was removed and 200 μl of each drug concentration or growth medium (controls) were distributed in the 96-well plates. Plates containing 5-FU, FUdR, and FUR were incubated for 24 h at 37°C. Oxaliplatin and CPT-11 were exposed to the cells in the other plates for 2 h, based on the length of time that has demonstrated clinical success (De Gramont et al. 2000; Saltz et al. 2000). Following incubation, the drugs were removed and each well was rinsed with growth medium. Wells in each plate then received 200 μl of fresh growth medium and cells were allowed to incubate for 2 days. At the end of the growth period, 50 μl of MTT (2 mg/ml) were added to each well and the cultures were incubated for 4 h at 37 °C in a humidified 5% CO2 atmosphere. The medium and MTT were removed from the wells and the remaining MTT-formazan crystals were dissolved by adding 200 μl of DMSO. After adding Sorensen’s glycine buffer (0.1 M glycine, 0.1 M NaCl adjusted to pH 10.5 with 1 M NaOH) to the wells containing DMSO, absorbance was measured with an ELISA plate reader at 570 nm (Dynex MRX Microplate Reader). Each experiment was performed using three replicated wells for each drug concentration and carried out independently three times. Results were expressed as percent cell survival. The percent cell survival for each drug concentration was calculated by the following formula: % survival = (absorbance of test wells/absorbance of control wells) × 100. A graph of % cell survival versus concentration for each drug was plotted to determine inhibitory concentrations (IC). The concentrations at which 90%, 70% and 30% of cells survived corresponded to IC10, IC30, and IC70, respectively. These values were determined for each drug based on their respective dose response graph.

Drug combination cytotoxicity assay

The MTT cytotoxicity assay was utilized to examine cell survival after particular combinations of oxaliplatin or CPT-11 with the fluoropyrimidines. After a 24 h incubation period, HT-29 cells were exposed for 2 h to IC10, IC30, and IC70 of oxaliplatin or CPT-11 (200 μl). The plates were incubated at 37 °C in a humidified 5% CO2 atmosphere. The drugs were then removed and the wells were rinsed with growth medium. These cells were subsequently exposed for 24 h to IC10, IC30, and IC70 of 5-FU, FUdR, and FUR. Every possible inhibitory concentration combination between the fluoropyrimidines and oxaliplatin or CPT-11 was represented. Cytotoxicity for each individual drug alone represented the controls. After removing the drug solutions from the wells and filling them with fresh medium, MTT was added. Percent cell survival was then determined based on the MTT assay described above. Samples were examined in triplicate and the experiment was repeated three times.

Cytotoxicity values from the various combinations were subjected to the multiple drug effect analysis of Chou and Talay (Chou and Talay 1987). Using the computer program CalcuSyn (Biosoft, Cambridge, UK), the Combination Index (CI) for every combination was computed. The CI indicated synergism when smaller than 0.8, antagonism when greater than 1.20, and additive cytotoxic effects when located between 0.80 and 1.20.

Fluoropyrimidine nucleic acid incorporation after pretreatment with oxaliplatin or CPT-11

Cytotoxic synergisms and antagonisms of particular combinations of oxaliplatin or CPT-11 with 5-FU, FUdR, or FUR should be explained by differences in nucleic acid incorporation. In each of 16 75-cm2 flasks, 3.5×106 HT-29 cells were seeded with 18 ml of growth medium for 2 days. Eight flasks were designated to receive oxaliplatin pretreatment and the remaining eight flasks contained cells that would not receive pretreatment (controls). After the 2-day incubation period, IC30 of oxaliplatin was added to each of the designated pretreatment flasks for 2 h. All flasks were then rinsed with medium and trypsinized. After pooling all of the cells from a particular group (oxaliplatin or control), cell counts were measured and the cell suspensions were centrifuged at 2,000 RPM for 5 min. Following the removal of the supernatant, the remaining cells were resuspended in transport buffer. Cells (105 μl) were distributed into 1.5-ml Eppendorf tubes and 45 μl of tritium-labeled fluoropyrimidine were then added for 1 h. IC30 and IC70 of [3H]5-FU, [3H]FUdR, and [3H]FUR represented the fluoropyrimidine concentrations that were added to the control and oxaliplatin pretreated cells. After the incubation period, all samples were centrifuged and washed with PBS (600 μl) for 5 min. All treated cells then received 5% cold PCA (500 μl). This step disintegrated the cell such that only nucleic acid incorporation of the tritium-labeled fluoropyrimidines remained. After waiting 30 min, samples were centrifuged at 2,000 RPM for 5 min and the supernatant was discarded. The remaining nucleic acids were dissolved in 0.5 M NaOH and later neutralized with 0.5 N HCl. Samples were transferred to vials, mixed with scintillation fluid, and counted for radioactivity. Each combination of oxaliplatin with IC30 and IC70 of all three fluoropyrimidines was examined in triplicate. The overall experiment was then repeated two separate times. Fluoropyrimidine nucleic acid incorporation was expressed as cpm/106 cells. Incorporation was compared between those cells receiving the IC30 and those receiving the IC70 of each of the tritium-labeled fluoropyrimidines.

The same procedure was utilized to evaluate fluoropyrimidine nucleic acid incorporation after pretreatment with IC30 CPT-11. Samples were examined in triplicate and the experiment was repeated twice. Fluoropyrimidine incorporation was then compared between those cells receiving the IC30 and those receiving the IC70 of each of the tritium-labeled fluoropyrimidines.

Statistical analyses

All data are presented as mean±SD. A one-way analysis of variance (ANOVA) was utilized to determine statistical significance among groups. t-tests with Bonferroni correction were used for individual contrasts within each group at a P< 0.05.

Results

Individual drug cytotoxicity

The MTT cytotoxicity assay revealed percent cell survival over a range of concentrations for oxaliplatin, CPT-11, 5-FU, FUdR, and FUR. From the dose response plots, the values for IC10, IC30, and IC70 for each of the drugs were interpolated and are displayed in Table 1.

Table 1.

Drug inhibitory concentrations (IC) determined by the MTT cytotoxicity assay on HT-29 cells. Fluoropyrimidines were exposed for 24 h, while oxaliplatin and CPT-11 were exposed for 2 h. Results are averages of three separate trials

Drug Inhibitory Concentrations (μM)
IC10 IC30 IC70
5-FU 0.083 0.95 9.6
FUdR 0.000355 0.00106 0.00918
FUR 0.00088 0.00815 0.0575
Oxaliplatin 0.0068 0.114 0.776
CPT-11 0.0835 1.374 25.21
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Drug combination cytotoxicity

After determining the inhibitory concentrations for each drug, combinations of oxaliplatin or CPT-11 with 5-FU, FUdR, or FUR were examined. The IC10, IC30, and IC70 of each of the drugs were combined to determine the most synergistic and antagonistic relationships. Table 2 illustrates percent survival data for every combination of oxaliplatin or CPT-11 with a fluoropyrimidine. After a particular oxaliplatin or CPT-11 pretreatment (i.e., IC10, IC30, and IC70), there were no survival differences seen between the fluoropyrimidines when their IC10, IC30, and IC70 data were specifically compared. The table also illustrates that pretreatment with IC10 oxaliplatin or CPT-11 did not result in synergy when combined with any fluoropyrimidine concentration (confirmed with a lack of CI <0.8). However, cytotoxic synergy was observed when IC30 of oxaliplatin and CPT-11 were combined with IC10 and IC30 of any of the fluoropyrimidines. These particular combinations all resulted in CI values less than 0.8 (IC30 oxaliplatin/IC10 and IC30 fluoropyrimidine CI between 0.70–0.79, IC30 CPT-11/IC10 and IC30 fluoropyrimidine CI between 0.58–0.78). Pretreatment with IC70 of oxaliplatin and CPT-11 was not improved by the addition of any IC value of a fluoropyrimidine. Cell survival remained at approximately 31% after IC70 oxaliplatin pretreatment, regardless of the type and concentration of fluoropyrimidine that was later added. After pretreatment with IC70 of CPT-11, the addition of IC10 and IC30 of a fluoropyrimidine did not offer superior cytotoxic results compared to giving IC70 of CPT-11 alone. Surprisingly, pretreating the cells with IC10 and IC30 of both oxaliplatin and CPT-11 resulted in antagonism when the IC70 of 5-FU, FUdR, and FUR were subsequently added. The exposure of IC70 of a fluoropyrimidine alone resulted in the survival of approximately 28% of the cells. However, pretreatment with IC10 and IC30 of oxaliplatin or CPT-11 combined with IC70 of a fluoropyrimidine resulted in cell survival ranging from 39% to 53%. This antagonism was confirmed with CI values that were all greater than 1.2 (IC10 and IC30 of oxaliplatin/IC70 fluoropyrimidine CI between 1.7–2.3, IC10 and IC30 of CPT-11/IC70 fluoropyrimidine CI between 2.0–3.3).

Table 2.

Percent survival of HT-29 cells after various combinations of oxaliplatin or CPT-11 with 5-FU, FUdR, and FUR (mean±SD). Results are averages of three separate trials. (Fluoropyrimidines—24 h exposure, Oxal=oxaliplatin pretreatment for 2 h, CPT=CPT-11 pretreatment for 2 h)

Drug Control Percent cell survival (%)
Oxal IC10 Oxal IC30 Oxal IC70 CPT IC10 CPT IC30 CPT IC70
Control 100 92.5±3.1 72.4±4.3 32.3±2.7 91.8±2.5 73.6±5.1 28.7±3.9
5-FU IC10 88.1±2.7 89.4±4.5 60.1±3.2* 31.4±3.9* 86.2±2.8 61.7±2.9* 22.9±3.6*
5-FU IC30 69.4±3.0 67.8±3.7 50.3±4.6* 31.1±1.2* 66.1±3.6 58.5±4.8* 22.1±2.2*
5-FU IC70 28.6±3.8 42.8±2.3* 41.1±4.2* 31.0±2.0 48.1±2.6* 39.8±4.1* 20.1±3.4
FUdR IC10 88.8±4.7 87.9±3.3 59.3±2.6* 33.5±2.9* 88.8±3.7 63±3.9* 23.5±1.7*
FUdR IC30 73.4±5.1 71.2±4.3 53.2±3.2* 32.4±3.8* 69.4±3.9 57.4±2.9* 21.4±1.8*
FUdR IC70 27.9±3.5 45.3±2.8* 42±4.1* 29.1±2.5 53.3±3.0* 41±3.6* 20.6±2.9
FUR IC10 91±4.8 88.3±3.7 61.1±4.3* 32.2±2.7* 88.5±4.2 64.1±5.7* 27.7±3.2*
FUR IC30 72.8±2.9 69.7±3.4 54.4±3.1* 31.9±3.9* 66.2±4.1 54.4±4.0* 24.2±2.6*
FUR IC70 28.8±3.2 46.7±3.6* 39.4±2.8* 30.2±3.1 50.9±4.2* 43.2±3.2* 21.3±2.9
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Utilizing Bonferroni multiple comparisons, * indicates a significant difference (P<0.05) in cell survival between a particular combination and the fluoropyrimidine control alone

The results that demonstrated unexpected antagonism prompted an exploration into possible mechanistic explanations. The difference between cytotoxicity achieved by the combination of IC30 oxaliplatin or CPT-11 with a fluoropyrimidine and the fluoropyrimidine alone decreased as the fluoropyrimidine IC increased (confirmed with increasing CI values). For example, a combination of IC30 oxaliplatin and IC10 5-FU resulted in 60.1% cell survival, a 28% cytotoxic improvement when compared to IC10 5-FU alone. The difference dropped to a 19% cytotoxic advantage after IC30 5-FU treatment. Cytotoxic improvement did not exist once IC70 5-FU was added. Instead, a 14.2% cell survival increase resulted when compared to IC70 5-FU alone.

Fluoropyrimidine nucleic acid incorporation after oxaliplatin or CPT-11 pretreatment

An examination of fluoropyrimidine nucleic acid incorporation resulted in different outcomes for the control group when compared to those cells receiving pretreatment. Table 3 and Table 4 illustrate that as the fluoropyrimidine inhibitory concentration increased from IC30 to IC70 for the control cells, nucleic acid incorporation of 5-FU, FUdR, and FUR also increased. In contrast, incorporation decreased for all three fluoropyrimidines in cells receiving oxaliplatin and CPT-11 pretreatment. The most significant incorporation difference existed within the 5-FU group. The increase from IC30 to IC70 resulted in a threefold reduction of 5-FU incorporation in the oxaliplatin pretreatment group (Table 3) and a sixfold decrease in the CPT-11 pretreatment group (Table 4).

Table 3.

Total nucleic acid incorporation of the fluoropyrimidines in HT-29 cells after pretreatment with oxaliplatin for 2 h (mean±SD). Results are averages of two separate trials

Total nucleic acid incorporation (cpm/106 cells)
Drug Control Oxaliplatin IC30
5-FU IC30 174±21.9(a) 269±29.3(b)
5-FU IC70 359±44.8(c) 87±8.9(d)
FUdR IC30 669±41.9(e) 1123±77.1(f)
FUdR IC70 1440±107.2(g) 957±51.1(h)
FUR IC30 2798±112.1(j) 4476±234.2(k)
FUR IC70 5421±319.3(m) 2073±156.3(n)
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ANOVA indicates significance with P<0.05 for all fluoropyrimidine treatment groups. Bonferroni multiple comparisons show this significance between a and b, c and d, e and f, g and h, j and k, m and n

Table 4.

Total nucleic acid incorporation of the fluoropyrimidines in HT-29 cells after pretreatment with CPT-11 for 2 h (mean±SD). Results are averages of two separate trials

Total nucleic acid incorporation (cpm/106 cells)
Drug Control CPT-11 IC30
5-FU IC30 131±31.7 170±39.2
5-FU IC70 268±50.6(a) 25±4.9(b)
FUdR IC30 627±49.3(c) 969±61.7(d)
FUdR IC70 1236±83.7(e) 825±74.9(f)
FUR IC30 2114±102.5(g) 2854±141.9(h)
FUR IC70 4273±271.5(j) 1821±99.2(k)
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ANOVA indicates significance with P<0.05 for all fluoropyrimidine treatment groups. Bonferroni multiple comparisons show this significance between a and b, c and d, e and f, g and h, j and k

Discussion

The combination of oxaliplatin or CPT-11 pretreatment with 5-FU, FUdR, and FUR yielded important results seen for the first time. After oxaliplatin and CPT-11 pretreatment, one fluoropyrimidine did not produce superior cytotoxicity over the others at IC10, IC30, and IC70. Thus, combining oxaliplatin or CPT-11 with FUdR or FUR may not result in superior clinical success when directly compared to a treatment arm consisting of oxaliplatin or CPT-11 combined with 5-FU. A comparison of the effect of oxaliplatin to CPT-11 for every fluoropyrimidine addition did not result in different cytotoxicity outcomes. Both oxaliplatin and CPT-11 are frequently entering many treatment protocols, and our study illustrates that one drug is not more cytotoxically effective over the other when combined with a fluoropyrimidine. Cytotoxic synergy was observed when IC30 oxaliplatin and CPT-11 were combined with IC10 and IC30 of all three fluoropyrimidines. Since pretreatment with IC10 oxaliplatin and CPT-11 did not improve cytotoxicity accomplished by the fluoropyrimidines alone, it can be concluded that a particular concentration of oxaliplatin and CPT-11 must be achieved to yield efficacious results.

Despite the synergy observed at particular pretreatment and fluoropyrimidine combinations, antagonism was also observed. The difference between cytotoxicity achieved by the combination of IC30 oxaliplatin or CPT-11 with a fluoropyrimidine and the fluoropyrimidine alone decreased as the fluoropyrimidine IC increased. Ultimately, the addition of IC70 of any fluoropyrimidine to oxaliplatin or CPT-11 resulted in cytotoxic antagonism when compared to the cytotoxicity achieved by IC70 of the fluoropyrimidine alone. Matsuoka et al. found similar in vitro results when combining CPT-11 with 5-FU (Matsuoka et al. 1995). The investigators demonstrated that as the 5-FU concentration increased, the ratio of the cytotoxicity attained from the combination to the cytotoxicity achieved by 5-FU alone decreased. In vitro resistance results from a step-wise increase in fluoropyrimidine concentration over a specific period of time. Sobrero et al. developed cell lines resistant to FUdR by such a method (Sobrero et al. 1985). The investigators demonstrated that impaired drug transport was the mechanism of resistance and not differences in the activities of thymidylate synthase, FUdR phosphorylase, 5’-Fluorouridine kinase, 5-fluorouridine phosphorylase, and 5-Fluorouracil phosphoribosyltransferase. Another study utilizing the H-9 cell line illustrated that drug influx and nucleoside accumulation were significantly decreased in those cells resistant to FUdR and FUR compared to the sensitive cells (Agarwal et al. 2001). These investigators also did not observe differences in the expression of thymidylate synthase and multidrug-resistant protein between parental and resistant cell lines. Pretreatment with oxaliplatin and CPT-11 combined with fluoropyrimidine addition does result in cytotoxic synergy at particular fluoropyrimidine inhibitory concentrations, but antagonism appears to progressively occur as the fluoropyrimidine concentration is increased up to IC70. This antagonism was then proposed to occur because of differences in cell transport or metabolism once a specific fluoropyrimidine concentration is attained. Perhaps, oxaliplatin and CPT-11 initially alter the transport or metabolism of the fluoropyrimidines and make it more difficult for the fluoropyrimidines to be later incorporated into nucleic acids at increasing concentrations.

An exploration into fluoropyrimidine influx and metabolism based on eventual nucleic acid incorporation led to many novel findings. Since a common transport inhibitor to all three fluoropyrimidines cannot be used, direct nucleic acid incorporation of the fluoropyrimidines was examined instead. Directly correlating with the cytotoxicity data, nucleic acid incorporation of the fluoropyrimidines decreased as their inhibitory concentrations increased to IC70 for those cells pretreated with oxaliplatin or CPT-11. As expected, nucleic acid incorporation increased when the fluoropyrimidine concentration increased to IC70 for the control cells that did not receive pretreatment. These results suggest that oxaliplatin or CPT-11 pretreatment does indeed inhibit fluoropyrimidine incorporation as the concentrations of the fluoropyrimidines increase. An inhibition of nucleic acid incorporation may reflect difficulties in cell transport and/or drug metabolism. The specific mechanism of action of oxaliplatin and CPT-11 could also be the cause of an inhibition of fluoropyrimidine nucleic acid incorporation. Oxaliplatin functions by forming interstrand and intrastrand cross-linking of DNA molecules. CPT-11 is a DNA topoisomerase I inhibitor. The direct effects that these agents have on DNA may make the incorporation of subsequent fluoropyrimidines more difficult as their concentrations increase. In our study, the most significant incorporation difference was seen for those cells receiving 5-FU. Since the combination of oxaliplatin or CPT-11 with 5-FU is currently being used in numerous treatment protocols, investigators must be aware of possible antagonisms if 5-FU concentration becomes exceedingly high or if it is used on a long-term basis.

Future experiments are being designed to determine what other mechanisms could be responsible for the antagonism observed at high fluoropyrimidine concentrations. Plasma membrane structure, expression of multidrug-resistance protein, expression of apoptotic markers, and measurements of catabolism all need to be compared before and after oxaliplatin or CPT-11 pretreatment. Our results indicate similar cytotoxicity for 5-FU, FUdR, and FUR when combined with oxaliplatin or CPT-11. To establish these findings clinically in terms of response rates, trials need to be conducted that involve treatment arms that directly compare 5-FU to the other fluoropyrimidines after oxaliplatin or CPT-11 pretreatment. Ultimately, our study offers insight into the possible effects of choosing alternative fluoropyrimidines to be combined with oxaliplatin or CPT-11 and the consequences associated with changing their concentrations.

References

  1. Agarwal RP, Han T, Fernandez M (2001) Reduced cellular transport and activation of fluoropyrimidine nucleosides and resistance in human lymphocytic cell lines selected for arabinosylcytosine resistance. Biochem Pharmacol 61:39–47 [DOI] [PubMed] [Google Scholar]
  2. Ansfield FJ, Curreri AR (1963) Further clinical comparison between 5-Fluorouracil (5-FU) and 5-fluoro-2’-deoxyuridine (5-FUDR). Cancer Chemother Rept 32:101–105 [PubMed] [Google Scholar]
  3. Arisawa Y, Sutanto-Ward E, Dalton RD, Sigurdson ER (1994) Short-term intrahepatic FUdR infusion combined with bolus mitomycin C: reduced risk for developing drug resistance. J Surg Oncol 56:75–80 [DOI] [PubMed] [Google Scholar]
  4. Boring CC, Squires TS, Tong T, Montgomery S (1994) Cancer statistics 1994. CA Cancer J Clin 44:7–26 [DOI] [PubMed] [Google Scholar]
  5. Buroker T, Samson M, Correa J, Fraile R, Vaitkevicius VK (1976) Hepatic artery infusion of 5-FUDR after prior systemic 5-fluorouracil. Cancer Treat Rep 60:1277–1279 [PubMed] [Google Scholar]
  6. Chou TC, Talay P (1987) Applications of the median-effect principle for the assessment of low-dose risk of carcinogens and for the quantitation of synergism and antagonism of chemotherapeutic agents. New avenues of development cancer chemotherapy. Bristol-Myers Symposium Series. Academic, New York, pp 37–64
  7. Cunningham D, Pyrhonen S, James RD, Punt CJ, Hickish TF, Heikkila R, Johannesen TB, Starkhammar H, Topham CA, Awad L, Jacques C, Herait P (1998) Randomised trial of irinotecan plus supportive care versus supportive care alone after fluorouracil failure for patients with metastatic colorectal cancer. Lancet 352:1413–1418 [DOI] [PubMed] [Google Scholar]
  8. De Gramont A, Figer A, Seymour M, Homerin M, Hmissi A, Cassidy J, Boni C, Cortes-Funes H, Cervantes A, Freyer G, Papamichael D, Le Bail N, Louvet C, Hendler D, de Braud F, Wilson C, Morvan F, Bonetti A (2000) Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer. J Clin Oncol 18:2938–2947 [DOI] [PubMed] [Google Scholar]
  9. Dolnick BJ, Pink JJ (1983) 5-fluorouracil modulation of dihydrofolate reductase RNA levels in methotrexate-resistant KB cells. J Biol Chem 258:13299–13306 [PubMed] [Google Scholar]
  10. Douillard JY, Cunningham D, Roth AD, Navarro M, James RD, Karasek P, Jandik P, Iveson T, Carmichael J, Alakl M, Gruia G, Awad L, Rougier P (2000) Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet 355:1041–1047 [DOI] [PubMed] [Google Scholar]
  11. Freshney RI (2000) Culture of animal cells: a manual of basic technique, 4th edn. Wiley-Liss, New York, pp 336–339 [Google Scholar]
  12. Geoffroy FJ, Allegra CJ, Sinha B, Grem JL (1994) Enhanced cytotoxicity with interleukin-1 alpha and 5-fluorouracil in HCT116 colon cancer cells. Oncol Res 6:581–591 [PubMed] [Google Scholar]
  13. Giacchetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, Chollet P, Llory JF, Letourneau Y, Coudert B, Bertheaut-Cvitkovic F, Larregain-Fournier D, Le Rol A, Walter S, Adam R, Misset JL, Levi F (2000) Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol 18:136–147 [DOI] [PubMed] [Google Scholar]
  14. Glazer RI, Lloyd LS (1982) Association of cell lethality with incorporation of 5-Fluorouracil and 5-Fluorouridine into nuclear RNA in human colon carcinoma cells in culture. Mol Pharmacol 9:468–473 [PubMed] [Google Scholar]
  15. Houghton JA, Houghton PJ, Wooten RS (1979) Mechanism of induction of gastrointestinal toxicity in the mouse by 5-fluorouracil, 5-fluorouridine, and 5-fluoro-2’-deoxyuridine. Cancer Res 39:2406–2413 [PubMed] [Google Scholar]
  16. Kessel D, Bruns R, Hall TC (1971) Determinants of responsiveness to 5-Fluorouridine in transplantable murine leukemias. Mol Pharmacol 7:117–121 [PubMed] [Google Scholar]
  17. Kufe DW, Major PP (1981) 5-Fluorouracil incorporation into human breast carcinoma RNA correlates with cytotoxicity. J Biol Chem 256:9802–9805 [PubMed] [Google Scholar]
  18. Langenbach RJ, Danenberg PV, Heidelberger C (1972) Thymidylate synthetase: mechanism of inhibition by 5-Fluoro-2’-Deoxyridylate. Biochem Biophys Res Commun 48:1565–1571 [DOI] [PubMed] [Google Scholar]
  19. Levi F, Misset JL, Brienza S, Adam R, Metzger G, Itzakhi M, Caussanel JP, Kunstlinger F, Lecouturier S, Descorps-Declere A (1992) A chronopharmacologic phase II clinical trial with 5-fluorouracil, folinic acid, and oxaliplatin using an ambulatory multichannel programmable pump. High antitumor effectiveness against metastatic colorectal cancer. Cancer 69:893–900 [DOI] [PubMed] [Google Scholar]
  20. Levi F, Zidani R, Brienza S, Dogliotti L, Perpoint B, Rotarski M, Letourneau Y, Llory JF, Chollet P, Le Rol A, Focan C (1999) A multicenter evaluation of intensified, ambulatory, chronomodulated chemotherapy with oxaliplatin, 5-fluorouracil, and leucovorin as initial treatment of patients with metastatic colorectal carcinoma. Cancer 85:2532–2540 [DOI] [PubMed] [Google Scholar]
  21. Levi FA, Zidani R, Vannetzel JM, Perpoint B, Focan C, Faggiuolo R, Chollet P, Garufi C, Itzhaki M, Dogliotti L (1994) Chronomodulated versus fixed-infusion-rate delivery of ambulatory chemotherapy with oxaliplatin, fluorouracil, and folinic acid (leucovorin) in patients with colorectal cancer metastases: a randomized multi-institutional trial. J Natl Cancer Inst 86:1608–1617 [DOI] [PubMed] [Google Scholar]
  22. Levi FA, Zidani R, Misset JL (1997) Randomised multicentre trial of chronotherapy with oxaliplatin, fluorouracil, and folinic acid in metastatic colorectal cancer. Lancet 350:681–686 [DOI] [PubMed] [Google Scholar]
  23. Levin RD, Gordon JH (1993) Fluorodeoxyuridine with continuous leucovorin infusion. A phase II clinical trial in patients with metastatic colorectal cancer. Cancer 72:2895–2901 [DOI] [PubMed] [Google Scholar]
  24. Link KH, Aigner KR, Peschau K, Warthona M, Schwemmle K, Danenburg PV (1988) Concentration and time dependence of the toxicity of fluorinated pyrimidines to HT 29 colorectal carcinoma cells. Cancer Chemother Pharmacol 22:58–62 [DOI] [PubMed] [Google Scholar]
  25. Mandel HG, Klubes P, Fernandes DJ (1979) Studies of the antitumor action of 5-Fluorouracil (FU). Bull Cancer 66:49–54 [PubMed] [Google Scholar]
  26. Matsuoka H, Yano K, Takiguchi S, Kono A, Seo Y, Saito T, Tomoda H (1995) Advantage of combined treatment of CPT-11 and 5-Fluorouracil. Anticancer Res 15:1447–1452 [PubMed] [Google Scholar]
  27. Peters GJ, Van Groeningen CJ, Laurensse EJ, Pinedo HM (1991) Thymidylate synthase from untreated human colorectal cancer and colonic mucosa: enzyme activity and inhibition by 5-fluoro-2’-deoxy-uridine-5’-monophosphate. Eur J Cancer 27:263–267 [DOI] [PubMed] [Google Scholar]
  28. Pinedo HM, Peters GF (1988) Fluorouracil: biochemistry and pharmacology. J Clin Oncol 6:1653–1664 [DOI] [PubMed] [Google Scholar]
  29. Richie JP (1992) Intravesical chemotherapy: treatment selection, techniques and results. Urol Clin North Am 19:521–527 [PubMed] [Google Scholar]
  30. Rougier P, Van Cutsem E, Bajetta E, Niederle N, Possinger K, Labianca R, Navarro M, Morant R, Bleiberg H, Wils J, Awad L, Herait P, Jacques C (1998) Randomised trial of irinotecan versus fluorouracil by continuous infusion after fluorouracil failure in patients with metastatic colorectal cancer. Lancet 352:1407–12 [DOI] [PubMed] [Google Scholar]
  31. Saltz LB, Cox JV, Blanke C, Rosen LS, Fehrenbacher L, Moore MJ, Maroun JA, Ackland SP, Locker PK, Pirotta N, Elfring GL, Miller LL (2000) Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. Irinotecan Study Group. N Engl J Med 343:905–914 [DOI] [PubMed] [Google Scholar]
  32. Sanguedolce R, Vultaggio G, Sanguedolce F, Modica G, Li Volsi F, Diana G, Guereio G, Bellanca L, Rausa L (1998) The role of thymidylate synthase levels in the prognosis and the treatment of patients with colorectal cancer. Anticancer Res 18:1515–1520 [PubMed] [Google Scholar]
  33. Schmoll HJ, Buchele T, Grothey A, Dempke W (1999) Where do we stand with 5-Fluorouracil? Semin Oncol 26:589–605 [PubMed] [Google Scholar]
  34. Shuey DL, Setzer RW, Lau C, Zucker RM, Elstein KH, Narotsky MG, Kavlock RJ, Rogers JM (1995) Biological modeling of 5-fluorouracil developmental toxicity. Toxicology 102:207–213 [DOI] [PubMed] [Google Scholar]
  35. Sobrero AF, Moir RD, Bertino JR, Handschumacher RE (1985) Defective facilitated diffusion of nucleosides, a primary mechanism of resistance to 5-Fluoro-2’-deoxyuridine in the HCT-8 human carcinoma line. Cancer Res 45:3155–3160 [PubMed] [Google Scholar]
  36. Song D, Weintjes MG, Gans Y, Au JL (1997) Bladder tissue pharmacokinetics and antitumor effect of intravesicle 5-fluorouridine. Clin Cancer Res 3:901–909 [PubMed] [Google Scholar]
  37. Sun D, Urrabaz R, Kelly S, Nguyen M, Weitman S (2002) Enhancement of DNA ligase I level by gemcitabine in human cancer cells. Clin Cancer Res 8:1189–1195 [PubMed] [Google Scholar]
  38. van Laar JA, Durrani FA, Rustum YM (1993) Antitumor activity of the weekly push schedule of 5-fluoro-2’-deoxyuridine ± N-phosphonacetyl-L-aspartate in mice bearing advanced colon carcinoma 26. Cancer Res 53:1560–1564 [PubMed] [Google Scholar]
  39. van Laar JA, Rustum YM, Ackland SP, van Groeningen CJ, Peters GJ (1998) Comparison of 5-Fluoro-2’-deoxyuridine with 5-Fluorouracil and their role in the treatment of colorectal cancer. Eur J Cancer 34:296–306 [DOI] [PubMed] [Google Scholar]
  40. Wilkinson DS, Pitot HC (1973) Inhibition of ribosomal ribonucleic acid maturation in Novikoff hepatoma cells by 5-fluorouridine. J Biol Chem 248:63 [PubMed] [Google Scholar]

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