The over-expression of thymidylate synthase (TS) and of the other folate cycle enzymes, is one of the major 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 (LSCQLYQR) showed cell growth inhibitory activity against two cisplatin-sensitive human ovarian cancer cell lines, 2008 and A2780 and their resistant counterparts, C13* (13-fold resistant to cDDP) and A2780/CP (10-fold resistant to cDDP) cell lines, respectively . Unlike 5-fluorouracil (5-FU), LR inhibition of TS activity was not accompained by the up-regulation of TS mRNA level1, which is an important mechanism of resistance to both cDDP and 5-FU [2]. The peptide significantly improved its activity when administrated with a suitable protein delivery system (Saint PhD, Synvolux Therapeutics, NL). To improve the intracellular delivery of LR, we designed a bioconjugate with folic acid (FA-LR) which enters the cells by exploiting the folate receptor alpha (FRα)-mediated endocytosis. Indeed, FRα is an attractive target for tumor-selective drug delivery, since it is a tumor-associated antigen that is over-expressed in greater than 90% of human OCs . On these basis we synthesized a selective N-terminal  conjugated folate-LR peptide (FA-LR, see figure). In fact, as reported in literature , the selective folate  conjugation is a crucial requirement for a fruitful folate receptor recognition of FA derived compounds. The tentative to introduce in a chemoselective manner a folate unit in N-terminal position of LR by solid phase peptide synthesis failed. Thus, the folic acid unit was assembled in solution reacting pteroic azide with [Glu0]-LR previously synthesized in solid phase. The structure of the final product was investigated and confirmed by mass spectrometry fragmentation experiments. In order to identify the most responsive potential targets for a FR-directed therapy, we assessed the FR expression levels in a panel of human OC cell lines, differently sensitive or resistant to cDDP. Our results indicate that IGROV-1 and OAW28 cell lines express the higher levels of both total and surface protein, while the TOV112D have the lowest. Substrate specificity studies showed that the folate bioconjugate FA-LR blocked competitively the binding of [3H]Folic acid to FR and consequently its cellular uptake, more in the former than in the latter cell lines, suggesting that FR is the effective substrate exploited by FA-LR for its internalization. When tested in vitro, FA-LR was found to specifically inhibit the growth of IGROV-1 and OAW28 cell lines in a dose-dependent manner, whereas cells exhibiting lower FRα expression (TOV112D) were less affected. Based on the high-selective property, the development of receptor mediated targeting therapy will be useful to improve the therapeutic efficacy and reduce side effects.

Designing a new bioconjugate FA-LR and studies of the effects in human ovarian cancer cell lines

PELA', Michela;TRAPELLA, Claudio;GUERRINI, Remo;
2013

Abstract

The over-expression of thymidylate synthase (TS) and of the other folate cycle enzymes, is one of the major 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 (LSCQLYQR) showed cell growth inhibitory activity against two cisplatin-sensitive human ovarian cancer cell lines, 2008 and A2780 and their resistant counterparts, C13* (13-fold resistant to cDDP) and A2780/CP (10-fold resistant to cDDP) cell lines, respectively . Unlike 5-fluorouracil (5-FU), LR inhibition of TS activity was not accompained by the up-regulation of TS mRNA level1, which is an important mechanism of resistance to both cDDP and 5-FU [2]. The peptide significantly improved its activity when administrated with a suitable protein delivery system (Saint PhD, Synvolux Therapeutics, NL). To improve the intracellular delivery of LR, we designed a bioconjugate with folic acid (FA-LR) which enters the cells by exploiting the folate receptor alpha (FRα)-mediated endocytosis. Indeed, FRα is an attractive target for tumor-selective drug delivery, since it is a tumor-associated antigen that is over-expressed in greater than 90% of human OCs . On these basis we synthesized a selective N-terminal  conjugated folate-LR peptide (FA-LR, see figure). In fact, as reported in literature , the selective folate  conjugation is a crucial requirement for a fruitful folate receptor recognition of FA derived compounds. The tentative to introduce in a chemoselective manner a folate unit in N-terminal position of LR by solid phase peptide synthesis failed. Thus, the folic acid unit was assembled in solution reacting pteroic azide with [Glu0]-LR previously synthesized in solid phase. The structure of the final product was investigated and confirmed by mass spectrometry fragmentation experiments. In order to identify the most responsive potential targets for a FR-directed therapy, we assessed the FR expression levels in a panel of human OC cell lines, differently sensitive or resistant to cDDP. Our results indicate that IGROV-1 and OAW28 cell lines express the higher levels of both total and surface protein, while the TOV112D have the lowest. Substrate specificity studies showed that the folate bioconjugate FA-LR blocked competitively the binding of [3H]Folic acid to FR and consequently its cellular uptake, more in the former than in the latter cell lines, suggesting that FR is the effective substrate exploited by FA-LR for its internalization. When tested in vitro, FA-LR was found to specifically inhibit the growth of IGROV-1 and OAW28 cell lines in a dose-dependent manner, whereas cells exhibiting lower FRα expression (TOV112D) were less affected. Based on the high-selective property, the development of receptor mediated targeting therapy will be useful to improve the therapeutic efficacy and reduce side effects.
2013
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2295822
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