Endocrine Abstracts

FSH receptor (FSHR) expression has been shown in gonadal tumours, as well as in endothelial tumour vessel cells of various cancers. FSHR, due to its transmembrane localization could be a good candidate for receptor-mediated targeted cancer therapy. In recent years, a number of membrane disrupting lytic peptides have been used for receptor-based cancer therapy. In the present study, we characterised the specificity and cytotoxicity of a lytic peptide Phor21 conjugated to three different amino-acid (AA) sequence fragments of the FSHβ-chain (AA33–53, AA81–95, and AA33–53+81–95) with several other enhancing modifications of the peptides, to ablate FSHR-positive cells in vitro. As there exists no cancer cell lines expressing functional endogenous FSHR, Phor21-FSHβ conjugates were tested in a human FSHR cDNA-stably transfected HEK293 cell line (FSHR-positive cell) and HEK293 (FSHR-negative cells). Conjugate-mediated cytotoxicity was assessed by measurement of lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon membrane disruption by lytic peptide. Conjugate of Phor21 linked to FSHβ–AA33–53, in which cysteine (Cys53) was replaced by serine (Ser) and stabilized with N-terminal amino group (Phor21-FSHβ33–53Cys/Ser-CONH) displayed the highest specific cytotoxicity to FSHR-positive cells vs. any other tested compound. Phor21-FSHβ33–53Cys/Ser-CONH conjugate showed in a dose dependent manner (0.5, 1, 2.5, and 5 μM) two- to ten fold higher ability to destroy the FSHR-positive cells than Phor21 alone. Pre-incubation or co-treatment competitive studies in FSHR-positive cells with recombinant human FSH 100 IU/L significantly decreased the cytotoxicity of Phor21-FSHβ33–53C/S-CONH conjugate at two lowest concentrations (0.25 and 0.5 μM). Conjugating FSHβ fragment sequences to Phor21 slightly enhanced their cytotoxic effects in comparison to Phor21 backbone in FSHR-negative cells. Our results prove the principle that Phor21-FSHβ33–53Cys/Ser-CONH may provide a novel specific therapeutic lead into targeted destruction of FSHR expressing cancer cells in vitro and as well as for further in vivo studies.

Disclosure: Polish National Science Centre (2012/05/B/NZ5/01867), Academy of Finland.