Studies on the cellular pharmacology of novel folic acid derivatives
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The main objective of this work was to determine the cytotoxicity of novel folic acid analogs designed in our laboratory, and to gain insight into the mechanism of their cellular uptake. The effects of different cell culture conditions on the inhibition of cell growth were also investigated. The two antifolates that were the subject of this study were VKN-62, an inhibitor of dihydrofolate reductase, and VKN-64, an inhibitor of thymidylate synthase. VKN-62 and VKN-64 are quinazoline derivatives of folic acid containing a unique indoline ring system. The positive controls chosen for this study were two cytotoxic antifolates: methotrexate (MTX), a prototype dihydrofolate reductase inhibitor; and N10-propargyl-5, 8-dideazafolate (CB-3717), a selective inhibitor of thymidylate synthase. Both prototype antifolates had been previously shown to differ in their mode of cellular entry. MTX is transported into mammalian cells primarily by the reduced-folate carrier (RFC). In contrast, CB3717 is internalized as a complex with the folate receptor-α (FR-α). In addition, 5-fluoro-2'-deoxyuridine (5-FdUrd) was also employed as a known inactivator of thymidylate synthase. The cell culture systems employed in these studies utilized 9L rat sarcoma and A2780 human ovarian carcinoma cell lines. Cell growth was measured colorimetrically, using either MTT (for studies in the 9L cell line) or SRB (for studies in the A2780 cell line) dyes. Although similar data were obtained with the two dyes, the SRB assay was used for the majority of the experiments because of its practical advantages. It was found that the possible presence of thymidine in the fetal bovine serum (FBS) added to the media interfered with the growth inhibitory effects of the thymidylate synthase inhibitors by decreasing the maximum achievable inhibition to about 50%. Likewise, the presence of folic acid in the RPMI 1640 media interfered with the effects of the antifolates. Therefore, dialyzed FBS, and media that was certified as "folate free" were employed in all subsequent experiments. It was hypothesized that the ring-modified folate derivatives, VKN-62 and VKN-64, are selective in their mode of entry into cells. VKN-62, like MTX, was expected to be transported into cells preferentially by the RFC, whereas VKN-64, like CB3717, was expected to enter cells preferentially via the FR-α. To test for the utilization of FR-α for cellular entry, 100 μM folic acid was added to medium to compete with the various antifolates, as an antagonist at the receptor. In the absence of folic acid, the IC50-values of VKN-64 and CB3717 were 7.0 x10 -7 M and 2.8 x 10 -7 M, respectively. In the presence of 100μM folic acid, the corresponding IC 50 -values were 7.7 x 10 -5 M and 6.6 x 10 -6 M, respectively. Thus, the presence of folic acid led to a 110-fold and a 24-fold decrease in potency of VKN-64 and CB3717 respectively, indicating a significant involvement of FR-α in their cellular uptake. In contrast, the IC 50 -values of VKN-62 and MTX were changed from 5.0 x 10 -8 M and 6.0 x 10 -9 M, to 4.0 x 10 -7 M and 6.4x10 -8 M respectively, upon addition of folic acid. This amounts to a difference of about 10-fold, suggesting that RFC is primarily responsible for the cellular entry of these antifolates. The slight reduction in their potencies may be accounted for by intracellular reversal of their effects by folic acid and its metabolites, independent of the FR-α. In as much as previous studies in our lab suggest that the novel antifolates are rationally designed to be more potent in their effect and more selective in their interaction with intracellular targets, further studies will be required to conclusively establish which mechanisms are responsible for their cellular uptake.