Limitations of inhibitory activities of polyphenols on furin-mediated substrate processing.

Recently, selected polyphenols were reported to exert proprotein convertase (PC) inhibitory activities on in vitro cleavage of a fluorogenic peptide substrate and it was concluded that this anti-protease activity might be responsible for the reported anti-cancer properties of these polyphenols. This prompted investigations to identify PC inhibiting polyphenols that could affect IGF-1R-mediated tumorigenesis since pro-IGF-1R is bioactivated by PCs like furin. Initial screening of polyphenols for their impact on in vitro cleavage of fluorogenic peptide substrate Pyr-RTKR-AMC by human furin (hfurin(573)) indeed revealed varying inhibitory effects. (-)EGCG, chrysin, and quercetin, were subsequently evaluated using uncleaved diphtheria toxin as substrate in vitro. However, none displayed any inhibitory impact on processing. Binding of (-)EGCG to both furin and the diphtheria toxin protein was demonstrated. Subsequently, it was found that for seven polyphenols tested, addition of casein or gamma globulin led to reduction or even annihilation of in vitro Pyr- RTKR-AMC cleavage inhibition. No such effect was seen with the furin inhibitor nona-D-arginine. Western blot studies to investigate possible effects of selected polyphenols on processing in cells of the tumorigenesis-linked proproteins pro-IGF-1R and pro-GPC3 also revealed no inhibitory effects. In conclusion, our results confirm the reported PC inhibitory effects of polyphenols on fluorogenic peptide substrate cleavage in vitro. However, the data show that polyphenolic inhibitory effects on hfurin(573)-mediated in vitro fluorogenic peptide substrate cleavage cannot be extrapolated to similar effects on processing of genuine proproteins, whether in vitro or in cells. This undermines the anti-protease rationale for the reported polyphenolic anti-cancer properties.