In vitro and in vivo antitumor activity of methotrexate conjugated to human serum albumin in human cancer cells

Clin Cancer Res. 2003 May;9(5):1917-26.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / therapeutic use*
  • Cell Cycle / drug effects
  • Cell Division / drug effects
  • Humans
  • In Vitro Techniques
  • Male
  • Methotrexate / therapeutic use*
  • Mice
  • Mice, Nude
  • Neoplasm Transplantation
  • Neoplasms / drug therapy*
  • Neoplasms / pathology
  • Serum Albumin / adverse effects
  • Serum Albumin / therapeutic use*
  • Thymidylate Synthase / antagonists & inhibitors
  • Thymidylate Synthase / metabolism
  • Transplantation, Heterologous
  • Tumor Cells, Cultured


  • Antineoplastic Agents
  • Serum Albumin
  • methotrexate-serum albumin
  • Thymidylate Synthase
  • Methotrexate