PTEROATE-PEPTIDE BIOCONJUGATE TARGETING THE FOLATE RECEPTOR IN HUMAN OVARIAN CANCER CELL LINES: TRANSPORT AND MECHANISM OF ACTION.

Objectives The over-expression of thymidylate synthase (TS) and of the other folate cycle enzymes, is one of the mechanisms of resistance to cisplatin (cDDP) encountered in most of resistant human ovarian cancer cell lines, accounting for the more efficient DNA repair and synthesis. Oligopeptides were designed to inhibit TS activity by interfering with its dimerization. Among these, the LR octapeptide showed cell growth inhibitory activity against two cisplatin-sensitive human ovarian cancer cell lines. To improve the intracellular delivery of LR, we designed a bioconjugate with folic acid (FA-LR), which enters cell by exploiting the folate receptor alpha (FRα)-mediated endocytosis. Methods -Cell lines. The human ovarian cancer cell lines OAW28, COV504, IGROV-1, TOV112D, 2008, C13*, A2780 and A2780/CP. -Real Time PCR of FRα mRNA . -Flow cytometric analysis of FRα cell surface expression -Folic acid surface binding studies. -Uptake studies Results Real Time PCR, western blot analysis and folic acid surface binding assay indicate that IGROV-1 and OAW28 cells show high expression levels of FRα, while TOV112D, 2008 and 2008/C13* almost don't express FRα on their cell surface. The folate bioconjugate FA-LR blocked competitively the binding of [3H]Folic acid to FR and consequently its cellular uptake. FA-LR is detected in the cell and its stability evaluated. Conclusions The chemical modification of the folate with the LR drug motif only minimally altered the intrinsic affinity the biocongiugate for FR and suggest that the pteroate-peptide conjugate exploits FR as a substrate for its internalization. Cytotoxicity of the bioconjugate will be presented.