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Pemetrexed (PMX, Alimta ®) is a new generation antifolate with activity in a variety of solid tumors. It is an excellent substrate for the reduced folate carrier (RFC) and folate receptor-α (FR-α). The role of FR-α in PMX pharmacological activity is uncertain. While high-level expression may enhance the activity of this agent, it is not clear as to what role constitutive levels of this transporter contribute to PMX activity. In this study, constitutive expression of FR-α was completely abolished by siRNA- induced silencing in HeLa cells and in RFC-null HeLa R5 cells as confirmed by Northern blotting, immuno-histochemistry, and cell surface specific [3H]folic acid binding. PMX growth inhibition over six days was unchanged in HeLa and R5 cells in the absence of FR-α expression. Loss of FR-α expression did not decrease net accumulation of 50 nM [3H]PMX over six days in either wild-type or RFC-null HeLa cells. Likewise, folate pools, measured after seven days growth in 25 nM [3H] 5-formyltetrahydrofolate, were not decreased by FR-α gene silencing in wild-type HeLa cells and were negligibly affected in the RFC-null R5 subline. Specific [3H]folic acid membrane binding in HeLa cells was compared to that of other human solid tumor cell lines. Although expression in HeLa cells was somewhat greater than in the OVCAR-6 and OVCAR-3 ovarian and CaCO-2 colon cancer cell lines, binding was more than ten-fold greater than in the majority of a panel of other human solid tumor cell lines of mesothelioma, hepatoma, breast, lung, and colon cancer origin. Hence, constitutively expressed FR-α in HeLa cells does not contribute to PMX activity in the presence or absence of RFC function. This is likely the case in many human solid tumor cell lines. This work was supported by grants from the National Institutes of Health (CA-82621) and the Eli Lilly Company.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]