Pemetrexed (
ALIMTA,
MTA) is a novel
thymidylate synthase (TS) inhibitor and has shown activity against
colon cancer,
mesothelioma and
nonsmall-cell lung cancer. We induced resistance to
Pemetrexed in the human
colon cancer cell line WiDr by using a continuous exposure to stepwise increasing
Pemetrexed concentrations (up to 20 microM) as well as a more clinically relevant schedule with intermittent exposure (up to 50 microM) for 4 hr every 7 days, resulting in WiDr variants WiDr-cPEM and WiDr-4PEM, respectively. However, using the same conditions, it was not possible to induce resistance in the WiDr/F cell line, a variant adapted to growth under low
folate conditions. Mechanisms of resistance to
Pemetrexed were determined at the level of TS,
folylpolyglutamate synthetase (FPGS) and
reduced folate carrier (RFC). WiDr-4PEM and WiDr-cPEM showed cross-resistance to the polyglutamatable TS inhibitor
Raltitrexed (6- and 19-fold, respectively) and the nonpolyglutamatable TS-inhibitor
Thymitaq (6- and 42-fold, respectively) but not to
5-fluorouracil. The ratios of TS
mRNA:
beta actin mRNA in WiDr-4PEM and WiDr-cPEM were 5-fold (P=0.01) and 18-fold (P=0.04) higher, respectively, compared to WiDr (ratio: 0.012). In addition, TS
protein expression in the resistant WiDr variants was elevated 3-fold compared to WiDr, while the catalytic activity of TS with 1 microM dUMP increased from 30 pmol/hr/10(6) cells in WiDr cells to 2201 and 7663 pmol/hr/10(6) cells in WiDr-4PEM and WiDr-cPEM, respectively. The activity of FPGS was moderately decreased, but not significantly different in all WiDr variants. Finally, no evidence was found that decreased catalytic activity of RFC was responsible for the obtained
Pemetrexed resistance. Altogether, these results indicate that resistance to
Pemetrexed in the
colon cancer cell line WiDr was solely due to upregulation of TS of which all related parameters (
mRNA and
protein expression and TS activity) were increased, rather than alterations in FPGS or RFC activity.