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1.
Nucleosides Nucleotides Nucleic Acids ; 27(6): 733-9, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18600533

ABSTRACT

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.


Subject(s)
Fluorouracil/metabolism , Fluorouracil/pharmacology , Leucovorin/pharmacology , RNA/metabolism , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/metabolism , Uridine/pharmacology , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cattle , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Mice , RNA/genetics , RNA Interference/drug effects , Sensitivity and Specificity
2.
Oncol Rep ; 19(6): 1517-23, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18497959

ABSTRACT

N-acetyl-dinaline (CI-994) is an investigational anti-cancer drug which inhibits histone deacetylases. We evaluated the interaction between CI-994 and conventional chemotherapeutics used in acute myeloid leukemia (AML) in a rat model for AML and Brown Norway rat acute myelocytic leukemia (BNML). In vitro, CI-994 in combination with cytarabine (ara-C), daunorubicin and mitoxantrone, resulted in moderate synergism. In vivo, higher dosages of CI-994 induced complete remissions. CI-994/ara-C was very active against BNML. The combinations of CI-994/daunorubicin and CI-994/mitoxantrone were also active against BNML. This study demonstrates favorable in vitro and in vivo interactions between CI-994 and conventional anti-cancer agents used for the treatment of AML.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Proliferation/drug effects , Leukemia, Myeloid, Acute/drug therapy , Phenylenediamines/administration & dosage , Animals , Benzamides , Blood Cell Count , Cytarabine/administration & dosage , Daunorubicin/administration & dosage , In Vitro Techniques , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Mitoxantrone/administration & dosage , Rats , Rats, Inbred BN
3.
Nucleosides Nucleotides Nucleic Acids ; 23(8-9): 1481-4, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15571281

ABSTRACT

A non-radioactive method to determine 5-Fluorouracil (5FU) incorporation into DNA has been developed. Isolated DNA was enzymatically degraded to bases and the resulting 5FU was measured with standard gas-chromatography coupled to mass spectrometry (GC-MS) and compared with that of radioactive 5FU in a cell line. Incorporation into DNA of the murine Colon 26-B tumor treated with maximal tolerated doses of 5FU and fluorodeoxyuridine (FUdR) was maximal after 2 hour and was 15.4 and 71.0 fmol/microg DNA, respectively. After a plateau for about 3 days a decrease was observed to +/- 2 fmol/microg DNA after 10 days. The assay is very sensitive and reproducible and can be used in a clinical setting.


Subject(s)
Antimetabolites, Antineoplastic/pharmacokinetics , DNA/drug effects , Drug Screening Assays, Antitumor/methods , Floxuridine/pharmacology , Fluorouracil/pharmacokinetics , Gas Chromatography-Mass Spectrometry/methods , Neoplasms/drug therapy , Animals , Cell Line, Tumor , Colonic Neoplasms/drug therapy , DNA/metabolism , Mice , Sensitivity and Specificity , Time Factors
4.
Ann Oncol ; 15(7): 1025-32, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15205195

ABSTRACT

BACKGROUND: The mechanism of action of 5-fluorouracil (5-FU) has been associated with inhibition of thymidylate synthase (TS) and incorporation of 5-FU into RNA and DNA, but limited data are available in human tumor tissue for the latter. We therefore measured incorporation in human tumor biopsy specimens after administration of a test dose of 5-FU alone or with leucovorin. PATIENTS AND METHODS: Patients received 5-FU (500 mg/m(2)) with or without high-dose leucovorin, low-dose leucovorin or l-leucovorin, and biopsy specimens were taken after approximately 2, 24 or 48 h. Tissues were pulverized and extracted for nucleic acids. 5-FU incorporation was measured using gas chromatography/mass spectrometry after complete degradation to bases of isolated RNA and DNA. RESULTS: Maximal incorporation into RNA (1.0 pmol/micrograms RNA) and DNA (127 fmol/micrograms DNA) of 59 and 46 biopsy specimens, respectively, was found at 24 h after 5-FU administration. Incorporation into RNA but not DNA was significantly correlated with intratumoral 5-FU levels. However, DNA incorporation was significantly correlated with the RNA incorporation. Primary tumor tissue, liver metastasis and normal mucosa did not show significant differences, while leucovorin had no effect. Neither for RNA (30 patients) nor DNA (24 patients) incorporation was a significant correlation with response to 5-FU therapy found. However, in the same group of patients, response was significantly correlated to TS inhibition (mean TS in responding and non-responding groups 45 and 231 pmol/h/mg protein, respectively; P=0.001). CONCLUSIONS: 5-FU is incorporated at detectable levels into RNA and DNA of human tumor tissue, but no relation between the efficacy of 5-FU treatment and incorporation was found, in contrast to TS.


Subject(s)
Colorectal Neoplasms/drug therapy , DNA, Neoplasm/metabolism , Fluorouracil/therapeutic use , RNA, Neoplasm/metabolism , Thymidylate Synthase/antagonists & inhibitors , Adult , Aged , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/metabolism , Antimetabolites, Antineoplastic/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Female , Fluorouracil/administration & dosage , Fluorouracil/metabolism , Hepatic Artery/injuries , Humans , Infusions, Intra-Arterial/methods , Leucovorin/administration & dosage , Male , Middle Aged , Thymidylate Synthase/metabolism , Time Factors , Treatment Outcome
5.
Br J Cancer ; 88(12): 1963-70, 2003 Jun 16.
Article in English | MEDLINE | ID: mdl-12799644

ABSTRACT

Gemcitabine (2',2'-difluorodeoxycytidine) is a deoxycytidine analogue that is activated by deoxycytidine kinase (dCK) to its monophosphate and subsequently to its triphosphate dFdCTP, which is incorporated into both RNA and DNA, leading to DNA damage. Multidrug resistance (MDR) is characterised by an overexpression of the membrane efflux pumps P-glycoprotein (P-gP) or multidrug resistance-associated protein (MRP). Gemcitabine was tested against human melanoma, non-small-cell lung cancer, small-cell lung cancer, epidermoid carcinoma and ovarian cancer cells with an MDR phenotype as a result of selection by drug exposure or by transfection with the mdr1 gene. These cell lines were nine- to 72-fold more sensitive to gemcitabine than their parental cell lines. The doxorubicin-resistant cells 2R120 (MRP1) and 2R160 (P-gP) were nine- and 28-fold more sensitive to gemcitabine than their parental SW1573 cells, respectively (P<0.01), which was completely reverted by 25 micro M verapamil. In 2R120 and 2R160 cells, dCK activities were seven- and four-fold higher than in SW1573, respectively, which was associated with an increased dCK mRNA and dCK protein. Inactivation by deoxycytidine deaminase was 2.9- and 2.2-fold decreased in 2R120 and 2R160, respectively. dFdCTP accumulation was similar in SW1573 and its MDR variants after 24 h exposure to 0.1 micro M gemcitabine, but dFdCTP was retained longer in 2R120 (P<0.001) and 2R160 (P<0.003) cells. 2R120 and 2R160 cells also incorporated four- and six-fold more [(3)H]gemcitabine into DNA (P<0.05), respectively. P-glycoprotein and MRP1 overexpression possibly caused a cellular stress resulting in increased gemcitabine metabolism and sensitivity, while reversal of collateral gemcitabine sensitivity by verapamil also suggests a direct relation between the presence of membrane efflux pumps and gemcitabine sensitivity.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Multidrug Resistance-Associated Proteins/metabolism , Neoplasms/metabolism , Cell Division/drug effects , DNA Damage , Deoxycytidine/metabolism , Humans , Neoplasms/drug therapy , Phosphorylation , Tumor Cells, Cultured , Gemcitabine
6.
Eur J Cancer ; 39(5): 691-7, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12628850

ABSTRACT

Deoxycytidine kinase (dCK) is required for the phosphorylation of several deoxyribonucleoside analogues that are widely employed as chemotherapeutic agents. Examples include cytosine arabinoside (Ara-C) and 2-chlorodeoxyadenosine (CdA) in the treatment of acute myeloid leukaemia (AML) and gemcitabine to treat solid tumours. In this study, expression of dCK mRNA was measured by a competitive template reverse transcriptase polymerase chain reaction (CT RT-PCR) in seven cell lines of different histological origin, 16 childhood and adult AML samples, 10 human liver samples and 11 human liver metastases of colorectal cancer origin. The enzyme activity and protein expression levels of dCK in the cell lines were closely related to the mRNA expression levels (r=0.75, P=0.026 and r=0.86, P=0.007). In AML samples, dCK mRNA expression ranged from 1.16 to 35.25 (x10(-3)xdCK/beta-actin). In the cell line panel, the range was 2.97-56.9 (x10(-3)xdCK/beta-actin) of dCK mRNA expression. The enzyme activity in liver metastases was correlated to dCK mRNA expression (r=0.497, P=0.05). In the liver samples, these were not correlated. dCK mRNA expression showed only a 36-fold range in liver while a 150-fold range was observed in the liver metastases. In addition, dCK activity and mean mRNA levels were 2.5-fold higher in the metastases than in the liver samples. Since dCK is associated with the sensitivity to deoxynucleoside analogues and because of the good correlation between the different dCK measurements in malignant cells and tumours, the CT-RT PCR assay will be useful in the selection of patients that can be treated with deoxycytidine analogues.


Subject(s)
Deoxycytidine Kinase/metabolism , Leukemia, Myeloid/enzymology , Liver Neoplasms/enzymology , Liver/enzymology , Adult , Biopsy , Blotting, Western , Child , Humans , Liver Neoplasms/secondary , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
7.
Biochim Biophys Acta ; 1587(2-3): 194-205, 2002 Jul 18.
Article in English | MEDLINE | ID: mdl-12084461

ABSTRACT

Thymidylate synthase (TS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), for which 5,10-methylene-tetrahydrofolate (CH(2)-THF) is the methyl donor. TS is an important target for chemotherapy; it is inhibited by folate and nucleotide analogs, such as by 5-fluoro-dUMP (FdUMP), the active metabolite of 5-fluorouracil (5FU). FdUMP forms a relatively stable ternary complex with TS and CH(2)THF, which is further stabilized by leucovorin (LV). 5FU treatment can induce TS expression, which might bypass dTMP depletion. An improved efficacy of 5FU might be achieved by increasing and prolonging TS inhibition, a prevention of dissociation of the ternary complex, and prevention of TS induction. In a panel of 17 colon cancer cells, including several variants with acquired resistance to 5FU, sensitivity was related to TS levels, but exclusion of the resistant variants abolished this relation. For antifolates, polyglutamylation was more important than the intrinsic TS level. Cells with low p53 levels were more sensitive to 5FU and the antifolate raltitrexed (RTX) than cells with high, mutated p53. Free TS protein down-regulates its own translation, but its transcription is regulated by E2F, a cell cycle checkpoint regulator. Together, this results in low TS levels in stationary phase cells. Although cells with a low TS might theoretically be more sensitive to 5FU, the low proliferation rate prevents induction of DNA damage and 5FU toxicity. TS levels were not related to polymorphisms of the TS promoter. Treatment with 5FU or RTX rapidly induced TS levels two- to five-fold. In animal models, 5FU treatment resulted in TS inhibition followed by a two- to three-fold TS induction. Both LV and a high dose of 5FU not only enhanced TS inhibition, but also prevented TS induction and increased the antitumor effect. In patients, TS levels as determined by enzyme activity assays, immunohistochemistry and mRNA expression, were related to a response to 5FU. 5FU treatment initially decreased TS levels, but this was followed by an induction, as seen with an increased ratio of TS protein over TS-mRNA. The clear retrospective relation between TS levels and response now forms the basis for a prospective study, in which TS levels are measured before treatment in order to determine the treatment protocol.


Subject(s)
Fluorouracil/pharmacology , Thymidylate Synthase/biosynthesis , Animals , Antimetabolites, Antineoplastic/metabolism , Antimetabolites, Antineoplastic/pharmacology , Drug Resistance, Neoplasm/physiology , Enzyme Induction/drug effects , Fluorouracil/metabolism , Folic Acid Antagonists/pharmacology , Humans , In Vitro Techniques , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/genetics , Polymorphism, Genetic , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/genetics , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism
8.
Cancer Res ; 61(9): 3675-81, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11325838

ABSTRACT

Plasma levels of folates and thymidine in mice are about 10-fold higher than in humans and may influence the therapeutic efficacy of thymidylate synthase (TS) inhibitors, such as 5-fluorouracil (5FU) and the antifolates pemetrexed (MTA) and raltitrexed (RTX). Therefore, we tested their therapeutic efficacy in various murine tumor models, grown in mice on a normal and a folate-depleted diet, with high and low thymidine kinase (TK) levels. MTA and RTX were inactive against Colon-26-10 [doubling times gained by treatment; growth delay factor (GDF), 0.5 and 0.3, respectively], whereas 5FU was very active (GDF, >10; complete cures). Colon-26-10/F, grown in mice on a folate-depleted diet, was more sensitive to RTX and MTA (GDF, 2.1 and 1.3, respectively) but not to 5FU (GDF, 1.2); however, leucovorin reversed the effect leading to cures. Folate depletion did not reverse resistance of Colon-26A and Colon-26G (low TK) to MTA and RTX, whereas leucovorin only enhanced the 5FU effect in Colon-26A and Colon-26A/F. Folic acid at 15 mg/kg did not improve the therapeutic efficacy of MTA in folate-deficient mice. The folate-depleted diet decreased the reduced folates in Colon-26A/F and Colon-26-G/F tumors less (4-5-fold; P < 0.01) than in Colon-26-10/F tumors (8-fold; P < 0.001). Folate depletion increased TS levels 2-3-fold in all of the models and TK levels 6-fold (P < 0.01) in Colon-26G/F, explaining the lack of activity of MTA and RTX in Colon-26G/F. In contrast, TK-deficient FM3A/TK tumors were much more sensitive to RTX, MTA, and 5FU than parent FM3A tumors, which have comparable TS levels. The rate of thymidine phosphorylysis varied considerably in all of the tumors without a clear relation to antitumor activity. In conclusion, tumor folates may potentiate (5FU) or protect (antifolates). Murine tumor models should combine low folates and low thymidine rescue to optimize preclinical testing of antifolates.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Enzyme Inhibitors/pharmacology , Folic Acid Antagonists/pharmacology , Folic Acid/metabolism , Guanine/analogs & derivatives , Thymidine/metabolism , Thymidylate Synthase/antagonists & inhibitors , Animals , Antimetabolites, Antineoplastic/administration & dosage , Cell Division/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Diet , Dose-Response Relationship, Drug , Drug Synergism , Female , Fluorouracil/administration & dosage , Fluorouracil/pharmacology , Folic Acid/administration & dosage , Folic Acid/blood , Glutamates/pharmacology , Guanine/pharmacology , Leucovorin/administration & dosage , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Pemetrexed , Quinazolines/pharmacology , Thiophenes/pharmacology , Thymidine/blood , Thymidine Kinase/metabolism , Thymidylate Synthase/metabolism
9.
Crit Rev Oncol Hematol ; 36(2-3): 141-57, 2000.
Article in English | MEDLINE | ID: mdl-11033303

ABSTRACT

New drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. In this paper, we review the characteristics and scope of a relatively simple cell-culture system with a three-dimensional organisation pattern - the multilayered postconfluent cell culture model. Solid tumour cell lines from diverse origins when grown in V-bottomed microtiter plates reach confluence in 3-5 days and then start to form multilayers. The initial exponential growth of the culture is followed by a plateau phase when cells reach confluence. This produces changes in the morphology of the cells. For some cell lines, it is possible to observe cell differentiation. A substantial advantage of the system is the use of the sulforodamine B (SRB) assay to determine relative cell growth or viability, which allows semiautomation of the experiments. Several experiments were performed to assess the differences and similarities between cells cultured as monolayers and multilayers, and eventually, compared with the results for solid tumours and some other models such as spheroids. Cell-cycle analysis for multilayers showed a lower S-phase arrest, which is accompanied by a decrease in the expression of cell-cycle-related proteins and a decrease in cellular nucleotide pools. Gene and protein expression of topoisomerase I, topoisomerase II and thymidylate synthase expression were lower for multilayers, but no substantial changes were observed for the expression of DT-diaphorase. P53 expression increased. Multilayer cultures present distinctive properties for drug transport across the membrane, drug accumulation and retention. In fact, the transport of antifolates across the membrane, accumulation of topotecan and gemcitabine-triphosphate are reduced in multilayers when compared with monolayers, which may be related to a decrease in drug penetration to the inner regions of the multilayers. Alteration of these pharmacodynamic parameters is directly related to a decrease in drug activity. The most powerful application of multilayers is in the assessment of cytotoxicity. Solid tumour cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The data show that multilayers are more resistant to the drugs than the corresponding monolayers, but there are substantial differences between the drugs depending on culture conditions, e.g. the difference was rather small for a drug such as cisplatin, miltefosine and EO9, a drug, which is activated under hypoxic conditions. Gemcitabine was active against ovarian cancer but not against colon cancer, resembling the in vivo situation. This observation was not evident with monolayer experiments. Another interesting application is the possibility to perform drug combination studies. The combination of gemcitabine and cisplatin proved to produce selective cell kill in H322 cells (non-small cell lung cancer cell line). Neither of the drugs was independently able to produce similar effects. In summary, multilayer cultures are relatively simple three-dimensional systems to study the effect of microenvironmental conditions on anticancer drug activity. The model might serve as a base for a more rigorous secondary in vitro screening.


Subject(s)
Cell Culture Techniques/methods , Drug Evaluation, Preclinical/methods , Animals , Cell Communication , Cell Division , Humans
10.
Pharmacol Ther ; 87(2-3): 227-53, 2000.
Article in English | MEDLINE | ID: mdl-11008002

ABSTRACT

Most current chemotherapy regimens for cancer consist of empirically designed combinations, based on efficacy and lack of overlapping toxicity. In the development of combinations, several aspects are often overlooked: (1) possible metabolic and biological interactions between drugs, (2) scheduling, and (3) different pharmacokinetic profiles. Antimetabolites are used widely in chemotherapy combinations for treatment of various leukemias and solid tumors. Ideally, the combination of two or more agents should be more effective than each agent separately (synergism), although additive and even antagonistic combinations may result in a higher therapeutic efficacy in the clinic. The median-drug effect analysis method is one of the most widely used methods for in vitro evaluation of combinations. Several examples of classical effective antimetabolite-(anti)metabolite combinations are discussed, such as that of methotrexate with 6-mercaptopurine or leucovorin in (childhood) leukemia and 5-fluorouracil (5FU) with leucovorin in colon cancer. More recent combinations include treatment of acute-myeloid leukemia with fludarabine and arabinosylcytosine. Other combinations, currently frequently used in the treatment of solid malignancies, include an antimetabolite with a DNA-damaging agent, such as gemcitabine with cisplatin and 5FU with the cisplatin analog oxaliplatin. The combination of 5FU and the topoisomerase inhibitor irinotecan is based on decreased repair of irinotecan-induced DNA damage. These combinations may increase induction of apoptosis. The latter combinations have dramatically changed the treatment of incurable cancers, such as lung and colon cancer, and have demonstrated that rationally designed drug combinations offer new possibilities to treat solid malignancies.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Fluorouracil/pharmacology , Neoplasms/drug therapy , Colonic Neoplasms/drug therapy , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine Kinase/metabolism , Drug Therapy/trends , Fluorouracil/administration & dosage , Humans , Leucovorin/pharmacology , Leukemia/drug therapy , Thymidylate Synthase/metabolism , Gemcitabine
11.
Int J Cancer ; 87(6): 771-8, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10956384

ABSTRACT

Cancer cell lines in standard cell culture medium or in animal models are surrounded by an environment with relatively high folate (HF) levels, compared with folate levels in human plasma. In the present study we adapted 4 colon cancer (C26-A, C26-10, C26-G and WiDr) and 3 squamous cell carcinoma of the head and neck (HNSCC) cell lines (11B, 14C and 22B) to culture medium with low folate (LF) levels (2.5, 1.0 and 0.5 nM, respectively) and investigated whether folate depletion had an effect on sensitivity to antifolates and which mechanisms were involved. All LF cell lines showed a higher sensitivity to 5-fluorouracil (5-FU) alone or in combination with leucovorin (LV) (2-5-fold), to the thymidylate synthase (TS) inhibitors, AG337 (2-7-fold), ZD1694 (3-49-fold), ZD9331 (3-40-fold), LY231514 (2-21-fold) or GW1843U89 (4-29-fold) or to the dihydrofolate reductase (DHFR) inhibitor PT523 (2-50-fold) compared with their HF variants cultured in standard medium containing up to 8 microM folic acid. LV could only increase sensitivity to 5-FU in HNSCC cell lines 14C and 14C/F. The differences in sensitivity could partially be explained by a 2-7-fold increased transport activity of the reduced folate carrier (RFC) in LF cell lines, whereas no significant change in folylpolyglutamate synthetase (FPGS) activity was observed. Furthermore, the protein expression and catalytic activity of the target enzyme TS were up to 7-fold higher in HF colon cancer cells compared with the LF variants (p < 0.05). Although the TS protein expression in LF HNSCC cells was also lower than in HF variants, the TS catalytic activity and FdUMP binding sites were up to 3-fold higher (p < 0.05). Thus, changes in TS levels were associated with differences in sensitivity. These results indicate that folate depletion was associated with changes in TS and RFC levels which resulted in an increase in sensitivity to 5-FU and antifolates. The folate levels in LF medium used in this study are more representative for folate levels in human plasma and therefore these data could be more predictive for the activity of 5-FU and antifolates in a clinical setting than results obtained from cell lines cultured in HF medium or in animal models.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Fluorouracil/pharmacology , Folic Acid Antagonists/pharmacology , Folic Acid/metabolism , Membrane Proteins , Membrane Transport Proteins , Neoplasms/drug therapy , Neoplasms/metabolism , Thymidylate Synthase/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/metabolism , Carrier Proteins/metabolism , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/metabolism , Humans , Leucovorin/therapeutic use , Lung Neoplasms , Mice , Multienzyme Complexes/antagonists & inhibitors , Peptide Synthases/metabolism , Reduced Folate Carrier Protein , Tetrahydrofolate Dehydrogenase , Thymidylate Synthase/antagonists & inhibitors , Tumor Cells, Cultured/drug effects
12.
Eur J Cancer ; 36(7): 916-24, 2000 May.
Article in English | MEDLINE | ID: mdl-10785598

ABSTRACT

Inhibition of the key enzyme in DNA synthesis, thymidylate synthase (TS), by 5-fluorouracil (5-FU) and the novel antifolate raltitrexed (Tomudex; ZD1694), induces dTTP depletion, resulting in DNA strand breaks, which can initiate pathways leading to an apoptotic mode of cell death. We studied 5-FU- and ZD1694-induced TS inhibition in relation to the expression of p53, p21, Bcl-2 and Bax in six colon carcinoma cell lines, two with a wild-type (wt) p53 (Lovo, LS174T) and four with a mutant (mt) p53 (WiDr, WiDr/F, HT29 and SW948) phenotype. In untreated cells, a reciprocal correlation between p53 and Bcl-2 was found: in cells with a low wt p53, Bcl-2 expression was present; whilst in cells with mt p53, Bcl-2 expression was not detectable. Exposure to 5-FU (50 and 100 microM) and ZD1694 (50 and 100 nM) for 24 and 48 h induced p53 and p21 expression in wt p53 cells, but not in mt p53 cells. TS was induced approximately 2-10-fold in all cell lines. TS induction was highest after ZD1694 exposure in the mt p53 cells HT29 and WiDr/F (6-10-fold). After 5-FU treatment, TS was present both as the free enzyme and in the ternary complex; however, predominantly as the ternary complex between TS, FdUMP and 5,10-methylenetetrahydrofolate. In wt p53 cells, both drugs increased Bax expression up to 5-fold, whereas in mt p53 cells, only a very slight induction was found. In wt p53 cells, Bcl-2 expression hardly changed after drug treatment. These results indicate a p53-independent induction of TS but a regulatory role of wt p53 in the synthesis of Bax in the colon carcinoma cell lines after TS inhibition.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colonic Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/genetics , Apoptosis , Blotting, Western , Colonic Neoplasms/enzymology , Enzyme Inhibitors/administration & dosage , Flow Cytometry , Fluorouracil/administration & dosage , Genes, bcl-2/genetics , Genes, p53/genetics , Humans , Quinazolines/administration & dosage , Thiophenes/administration & dosage , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/genetics , Thymidylate Synthase/metabolism , Tumor Cells, Cultured , bcl-2-Associated X Protein
15.
J Cancer Res Clin Oncol ; 125(12): 675-84, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10592100

ABSTRACT

The aim of the present studies was to elucidate the effects and optimal modulatory conditions of 5-ethyl-2'-deoxyuridine (EtdUrd) on the antitumour efficacy, pharmacokinetics and catabolism of 5-fluorouracil (5-FU) on Colon-26 and Colon-38 murine tumours. HPLC and GC-MS techniques were used to measure the concentrations of 5-FU, dihydro-5-fluorouracil, EtdUrd, 5-ethyluracil and uridine in the plasma and that of 5-FU and 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) in the tumours. It was shown that EtdUrd, given 1 h before 5-FU, selectively enhanced the antitumour action of 5-FU, without significantly increasing its toxic side-effects, thus resulting in an approximately three times higher therapeutic index. Pharmacokinetic studies revealed that 1 h after 400 mg/kg EtdUrd administration - i.e. at the time of 5-FU treatment - the plasma concentration of EtdUrd was 269 microM, and that of 5-ethyluracil, as the major metabolite of EtdUrd, was 421 microM. It is of interest that EtdUrd pretreatment did not change the maximal plasma concentration of 5-FU; however, the half-life of the terminal elimination increased from 114.5 min to 171.2 min and thus the mean residence time of 5-FU rose significantly (P < 0.05). After the combined treatment, the maximal concentration of dihydro-5-fluorouracil in the plasma decreased from 61.06 microM to 29.70 microM (P < 0.01). The intratumoral concentrations of 5-FU were 34%-158% higher 6-96 h after the combined treatment than after the single 5-FU treatment. EtdUrd also caused a moderate increase in the intratumoral level of FdUMP. It is noteworthy, that EtdUrd increased the endogenous uridine concentration in the plasma from 18 microM to a maximum of 249 microM, and the level remained high for longer than 6 h. The present studies indicate that EtdUrd enhances the therapeutic index of 5-FU by reducing the catabolism, prolonging the plasma and intratumoral concentrations of 5-FU and, at the same time, offering protection to normal organs by increasing the endogenous uridine level.


Subject(s)
Antimetabolites, Antineoplastic/pharmacokinetics , Colonic Neoplasms/drug therapy , Deoxyuridine/analogs & derivatives , Fluorouracil/pharmacokinetics , Animals , Antimetabolites, Antineoplastic/therapeutic use , Area Under Curve , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Deoxyuridine/blood , Deoxyuridine/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Fluorodeoxyuridylate/metabolism , Fluorouracil/analogs & derivatives , Fluorouracil/blood , Fluorouracil/therapeutic use , Male , Metabolic Clearance Rate , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Time Factors , Treatment Outcome , Uracil/analogs & derivatives , Uracil/blood , Uridine/blood
16.
Anticancer Drugs ; 10(5): 445-52, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10477163

ABSTRACT

Both gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum) have significant anticancer activity against ovarian, head and neck, and non-small cell lung cancer (NSCLC). dFdC can be incorporated into DNA and RNA, and inhibit DNA repair, while cisplatin can form Pt-DNA adducts. We previously observed schedule-dependent synergism of the combination of dFdC and cisplatin in monolayer cell cultures. We now evaluated whether the combination would also enable selective cell kill in multilayered postconfluent cell cultures, since each compound showed variable activity in multilayered cells. The combination was tested in multilayered cultures from cell lines with a different histological origin: the human head and neck squamous cell carcinoma cell line UMSCC-22B (22B), the human NSCLC cell line H322, and ADDP, a cisplatin-resistant variant of the human ovarian cancer cell line A2780. Sensitivity of the multilayered cells was dependent on exposure duration and sequence of the drug combinations, which were added in a constant molar ratio (dFdC:cisplatin 1:100), with a total exposure time of 96 h. The type of interaction was related to the degree of resistance of the cell lines to either dFdC or cisplatin. Thus, the very sensitive 22B cells only showed an additive effect when cells were preincubated for 24 h with dFdC prior to exposure to the combination. In contrast, in the resistant ADDP and H322 cells, synergism was the most common profile (three out of four schedules tested). This is of special relevance when we take into account that these drugs only show cytostatic effects when administered alone, whereas the combination produced cytotoxic cell killing. In conclusion, combining dFdC with cisplatin can be at least additive, but synergistic in multilayered postconfluent cells resistant to dFdC and cisplatin.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cisplatin/administration & dosage , Deoxycytidine/analogs & derivatives , Cell Survival/drug effects , Deoxycytidine/administration & dosage , Dose-Response Relationship, Drug , Drug Synergism , Humans , Tumor Cells, Cultured , Gemcitabine
17.
Nucleosides Nucleotides ; 18(4-5): 877-8, 1999.
Article in English | MEDLINE | ID: mdl-10432699

ABSTRACT

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5'-position with fatty acids (16-22 C-atoms, 0-3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cytarabine/analogs & derivatives , Animals , Antimetabolites, Antineoplastic/chemistry , Cytarabine/chemistry , Cytarabine/pharmacology , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Mice , Rats , Structure-Activity Relationship , Tumor Cells, Cultured , Gemcitabine
18.
Clin Cancer Res ; 5(3): 643-54, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10100718

ABSTRACT

Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. In a panel of 13 nonselected human colon cancer cell lines, we evaluated the role of TS levels in sensitivity to 5-fluorouracil (5FU) and four folate-based TS inhibitors that have been introduced recently into the clinic: ZD1694 (Tomudex, Raltitrexed, TDX), GW1843U89 (GW), LY231514 (LY), and AG337 (Thymitaq, AG). Because the latter compounds have different transport and polyglutamylation characteristics, we also related these parameters with drug sensitivity, measured by the sulforhodamine B assay after 72 h of drug exposure. For 5FU, the IC50s varied from 0.8 to 43.0 microM. Leucovorin (LV) potentiated the activity of 5FU in only 4 of 13 cell lines. Sensitivity to folate-based TS inhibitors was variable; IC50s were in the range of: 5.3-59.0 nM TDX; 11.0-1570 nM LY; and 0.5-8.9 nM GW. Eleven of 13 cell lines had an IC50 for AG between 1.3 and 5.3 microM. Two cell lines were resistant to AG, Colo201 and SW1116, with IC50s of 27 and 29 microM, respectively. TS catalytic activity (conversion of dUMP to dTMP) varied from 62 to 777 pmol/h/10(6) cells. The number of FdUMP binding sites varied from 32 to 231 fmol/10(6) cells. Regression analysis showed a significant relation between TS catalytic activity and IC50s for 5FU and 5FU/LV. Kis for FdUMP showed a significant Spearman rank correlation with the IC50s of AG and GW. The role of antifolate transport, accumulation, and polyglutamylation was determined with [3H]methotrexate (MTX) as a reference compound. [3H]MTX influx via the reduced folate carrier varied from 18.6 to 150 fmol/10(6) cells/min. Folylpolyglutamate synthetase (FPGS) activity showed a range from 47 to 429 pmol/10(6) cells/h. A total of 24 h of [3H]MTX accumulation showed a 20-fold variation, from 1.2 to 21.8 pmol/10(6) cells. FPGS levels showed a Spearman rank positive correlation with cytotoxicity to TDX. In conclusion, in a heterogeneous nonselected human colon cancer cell line panel, the best predictor for sensitivity to 5FU and 5FU/LV was TS activity. Multiple sensitivity determinants were of importance for antifolate TS inhibitors, including FPGS activity and TS enzyme kinetics.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Colonic Neoplasms/enzymology , Fluorouracil/pharmacology , Pteroylpolyglutamic Acids/metabolism , Thymidylate Synthase/metabolism , Antimetabolites, Antineoplastic/metabolism , Binding Sites , Biological Transport , Blotting, Western , Catalysis , Cell Division/drug effects , Colonic Neoplasms/pathology , Drug Screening Assays, Antitumor , Fluorodeoxyuridylate/metabolism , Fluorouracil/metabolism , Folic Acid Antagonists/pharmacology , Humans , Peptide Synthases/metabolism , Thymidylate Synthase/antagonists & inhibitors , Tritium , Tumor Cells, Cultured
19.
Oncol Res ; 11(8): 383-91, 1999.
Article in English | MEDLINE | ID: mdl-10803742

ABSTRACT

The combined cytotoxic effects of the thymidylate synthase (TS) inhibitors 5-fluorouracil (5FU) and different antifolates were studied in seven colon cancer cell lines. Growth inhibition of the antifolates, Nolatrexed, Raltitrexed, GW1843U89, or MTA in combination with 5FU, was determined and multiple drug effect analysis showed that the drugs acted mostly additively. The only synergistic interaction was found for 5FU and Nolatrexed in the LS174T cell line. Also Raltitrexed and 5FU were slightly synergistic in WiDr/F cells grown at low folate levels, but for the other cell lines grown at high folate levels this combination was more antagonistic. GW1843U89 and 5FU were mainly additive, while 5FU and MTA showed antagonism in WiDr and additivity in LS174T. The effect of the drugs at their target was evaluated by in situ TS inhibition. We observed lower TS activity in all cells when two drugs were used instead of one. Statistical analysis revealed that none of the values of the combinations was higher or lower than could be expected from the product of the effect of single drugs. We concluded that the effects on TS inhibition were additive for all 5FU/antifolate combinations in all cell lines. DNA strand break formation, as a result of TS inhibition, was measured by means of a fluorometric analysis of DNA unwinding. Raltitrexed-induced DNA damage was significantly increased by 5FU in WiDr cells [single agent: 67% double stranded (ds) DNA, combination: 39% ds DNA, P<0.0001]. In LS174T a trend for antagonistic effects was observed for combinations of MTA, GW1843U89, or Raltitrexed and 5FU. The combinations showed additive effects in WiDr/F cells. The overall conclusion of the three assays in each of the cell lines indicated that 5FU and antifolate combinations were predominantly additive in colon cancer cells.


Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Colonic Neoplasms/drug therapy , Fluorouracil/administration & dosage , Folic Acid Antagonists/administration & dosage , Cell Division/drug effects , Colonic Neoplasms/pathology , DNA Damage/drug effects , Glutamates/administration & dosage , Guanine/administration & dosage , Guanine/analogs & derivatives , Humans , Indoles/administration & dosage , Isoindoles , Pemetrexed , Quinazolines/administration & dosage , Thiophenes/administration & dosage , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/metabolism , Tumor Cells, Cultured
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