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.