Reduced, Nonprotein Thiols Inhibit Activation and Function of MMPs

Matrix metalloproteinase (MMP) latency is attributable to a complex formed between a reduced cysteine residue in the propeptide domain and the Zn2+ ion at the enzyme's catalytic site. Subsequently, an oxidation in the local redox state activates MMPs. Despite the obvious importance of this reduced thiol in MMP regulation, limited studies have assessed the effects of nonprotein thiols on MMP activation and function. Objectives: This study evaluated the effects of two nonprotein thiols, glutathione and N-acetylcysteine, on proMMP-2 and proMMP-9 activation, and mature enzyme gelatinase function. Methods: The abilities of glutathione and N-acetylcysteine to suppress proMMP activation (gelatin zymography), inhibit mature enzyme activities (fluorigenic substrate assay) and tumor cell basement membrane invasion (in vitro cell invasion assay) were determined. Results: Gelatin zymography demonstrated that inclusion of either glutathione or N-acetylcysteine (but not oxidized glutathione, GSSG) abolished proMMP-2 and proMMP-9 activation by the organomercurial compound APMA. Furthermore, fluorigenic substrate assays showed that glutathione and N-acetylcysteine (not GSSG) significantly inhibited MMP-9's gelatinase function (n=5, p<0.05, Yates corrected Chi square test). While both compounds also suppressed MMP-2 function, these reductions were not significant. Although N-acetylcysteine pretreatment suppressed cancer cell invasion, only a combination treatment (25 mM N-acetylcysteine+10 µg/ml endostatin) significantly inhibited invasion of oral squamous cell carcinoma cell lines (n=8, p<0.005, Yates corrected Chi square). Notably, SCC cell lines were obtained from different human donors. Studies are ongoing to determine whether a dose-dependent MMP inhibitory effect is present and to clarify the nature of the glutathione/N-acetylcysteine MMP-9 interactions. Conclusions: As MMP dysfunction contributes to the pathogenesis of numerous human diseases, identification of MMP regulating agents is a therapeutic research priority. Our data, which demonstrate that glutathione and N-acetylcysteine suppress proMMP activation, inhibit active MMP function, and reduce cancer cell invasion, introduce the potential to use these naturally-occurring compounds as MMP inhibitors. NIH/NCI-CA-RO195901(Mallery).