Abstract
We examined the importance of dosing interval between leucovorin (LCV) and 5-fluorouracil (5-FU) on intracellular thymidylate synthase (TS) ternary complex, free TS and total TS protein levels in human MCF-7 breast and NCI H630 colon cancer cell lines. A 2- to 3-fold increase in total TS was noted when either cell line was exposed to 5-FU 10 microM plus LCV (0.01-10 microM) compared with a 1.4- to 1.6-fold increase in total TS due to 5-FU 10 microM alone. The amount of TS ternary complex formed was 2- to 3-fold higher in both cell lines treated with the combination of 5-FU and LCV compared with 5-FU alone. TS complex formation and total TS protein increased with LCV dose (0.1-10 microM). In MCF-7 cells, the maximal increase in total TS protein and TS ternary complex formation was observed when 5-FU was delayed for 4 h after the start of LCV exposure. In NCI H630 cells, maximal total TS protein and ternary complex formation occurred when 5-FU was delayed for 18 h after the start of LCV exposure. The amount of free TS did not change in either cell line whether 5-FU was given concurrently with LCV or delayed for up to 24 h. The accumulation rate of intracellular folates in the form of higher glutamates Glu3-Glu5 was rapid in MCF-7 cells (maximal formation after 4 h), whereas in H630 cells accumulation of higher polyglutamates continued to increase up to 18 h. The time of peak folate polyglutamate (Glu3-Glu5) formation coincided with the time of peak TS complex formation and total TS protein in each cell line. In these human carcinoma cell lines, the LCV dose and interval between 5-FU and LCV play a role in increased TS total protein and TS ternary complex; however, the amount of free TS is independent of the interval between 5-FU and LCV. The time-and dose-dependent increases in TS ternary complex and TS total protein are associated with differences in the accumulation of folate polyglutamates in these cell lines.
Highlights
MCF-7 breast and NCI H630 colon cancer cells were exposed to LCV concentrations ranging from 0.1 to 1O JtM for 4 h
The effect of the 5-FU LCV schedule upon thymidylate synthase (TS) ternary complex formation was examined for an interval of 24 h from the start of LCV exposure using concentrations of 5-FU LCV that were non-growth inhibitory (5-lO% growth inhibition)
In MCF-7 cells. the maximal TS ternary complex formation occurred when 5-FU was delayed for 4 h after the start of LCV administration (Figure 3a). This resulted in a 1.8± 0.2-fold increase in TS ternary complex to free TS ratio over that seen when both drugs were added simultaneously (Figure 3a)
Summary
The goal of the present study was to determine the importance of the dose and interval of exposure between LCV and 5-FU on intracellular TS ternary complex formation and TS protein levels in human colon and breast cancer cell lines
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