To avoid systemic toxicity of the cytotoxic
drug methotrexate (MTX) and to improve
tumor selectivity, MTX was bound to
human serum albumin (HSA) as a
drug carrier. To understand more about the mechanism of action of MTX conjugated to HSA (
MTX-HSA), the uptake of
MTX-HSA into the cell was determined as well as the effect of
MTX-HSA on
thymidylate synthase (TS), cell cycle distribution, and cell proliferation. Different uptake kinetics were observed for [(3)H]MTX and [(3)H]
MTX-HSA. However, similar uptake kinetics were measured for (125)I-HSA and (125)I-MTX-HSA (2.1 and 1.8 pmol/10(7) cells/h when cells were treated with 10 micro M (125)I-HSA and (125)I-
MTX-HSA, respectively), suggesting that
MTX-HSA enters the cells by
albumin-mediated endocytosis. We observed no effect of
MTX-HSA on TS when
folate receptor-expressing KB cells were treated for 4 h (IC(50), >50 micro M). However, 24 h after incubation,
MTX-HSA inhibited TS with an IC(50) of 6.9 micro M. In addition, we found that
MTX-HSA had a delayed effect on the cell cycle compared with MTX and that this effect could be inhibited with the lysosomal inhibitor
methylamine, suggesting that
MTX-HSA activity is dependent on lysosomal processes. The proliferation of different wild-type and MTX-resistant tumor cell lines was inhibited at IC(50) concentrations between 2 and 78 micro M, respectively.
MTX-HSA accumulates in vivo in the
tumor tissue. Local concentrations of 18-29 micro M were measured, which are effective antiproliferative concentrations as determined in vitro. We also investigated the antitumor activity of
MTX-HSA in vivo in different human
tumor xenografts grown s.c. in nude mice. Fourteen
tumors from eight different tissues were tested. Nine of 14
tumors (64%) showed a clear response with
tumor inhibition, stasis, or regression; 5 of 14 (36%) gave a moderate response with
tumor growth delay or no response. In conclusion,
MTX-HSA is effectively taken up by the cells via
albumin receptor- or
folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations. In vivo, effective concentrations of
MTX-HSA were reached in
tumor tissue to exhibit antitumor activity.