Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells.
5-Fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FdUrd) and 5-trifluorothymidine (F3(d)Thd) are antimetabolites which are metabolized to their corresponding active forms which inhibit DNA synthesis via inhibition of thymidylate synthase (TS). To investigate ways of overcoming 5-FU-resistance, we established acquired-resistant colorectal cancer cell lines against these three drugs by continuous and step-wise escalation of drugs, and analyzed the cytotoxicity and the mechanism of resistance to the drugs. When cells were incubated with the 3 drugs for 72 h, the resistance ratio to parental DLD-1 human colorectal tumor cells was 65.2 for DLD-1/5-FU, 9.7 for DLD-1/FdUrd and 448.6 for DLD-1/F3(d)Thd cells. DLD-1/5-FU cells did not show any cross-resistance against FdUrd and F(3)dThd. However, DLD-1/FdUrd cells showed 3- and 9-fold increased resistance to 5-FU and F3(d)Thd, respectively, and DLD-1/F3(d)Thd cells also showed about 90-fold resistance to FdUrd. Analysis of enzyme activities and gene expression associated with pyrimidine metabolism indicated that a significant decrease in orotate phosphoribosyltransferase activity in DLD-1/5-FU cells, a 7-fold increase of TS mRNA in DLD-1/FdUrd cells, and a 37-fold decrease in thymidine kinase activity of DLD-1/F3(d)Thd cells were the major mechanisms of drug resistance. These findings were closely associated with the cytotoxicity of 5-FU, FdUrd and F3(d)Thd against the established 5-FU-, FdUrd- or F3(d)Thd-resistant cells. When DLD-1/FdUrd cells expressing increased TS mRNA were treated with FdUrd and F3(d)Thd for only 4 h, the resistance ratios of DLD-1/FdUrd cells to parental DLD-1 cells were markedly different for FdUrd and F3(d)Thd, suggesting that the cytotoxicity with short-time exposure to F3(d)Thd is due to a mechanism other than TS inhibition, although the cytotoxicity of F3(d)Thd in the short-time is low compared to that of long-time exposure. In conclusion, F3(d)Thd, an antimetabolite that inhibits TS activity, may be effective against 5-FU and/or FdUrd-resistance in colorectal cancer cells caused by amplification of TS and/or deletion of orotate phosphoribosyltransferase.
Murakami
Y
Y
The Second Cancer Laboratory, Taiho Pharmaceutical Co. Ltd., Hanno-city, Saitama 357-8527, Japan.
Kazuno
H
H
Emura
T
T
Tsujimoto
H
H
Suzuki
N
N
Fukushima
M
M
D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics* / Q000627:therapeutic use; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005467:Floxuridine / Q000493:pharmacokinetics* / Q000627:therapeutic use; D005472:Fluorouracil / Q000493:pharmacokinetics* / Q000627:therapeutic use; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism; D014271:Trifluridine / Q000493:pharmacokinetics* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D009363:Neoplasm Proteins; D005467:Floxuridine; D013940:Thymidylate Synthase; D014271:Trifluridine; D005472:Fluorouracil