Elsevier

Free Radical Biology and Medicine

Volume 41, Issue 8, 15 October 2006, Pages 1315-1324
Free Radical Biology and Medicine

Original Contribution
Reduced nonprotein thiols inhibit activation and function of MMP-9: Implications for chemoprevention

https://doi.org/10.1016/j.freeradbiomed.2006.07.014Get rights and content

Abstract

Clinical studies demonstrate a positive correlation between the extent of matrix metalloproteinase (MMP) activation and malignant progression of precancerous lesions. Therefore, identification of effective, well-tolerated MMP inhibitors represents a rational chemopreventive strategy. A variety of agents, including proteinases and thiol-oxidizing compounds, activate MMPs by initiating release of the propeptide's cysteine sulfur “blockage” of the MMP active site. Despite the importance of the propeptide's cysteine thiol in preserving MMP latency, limited studies have evaluated the effects of reduced thiols on MMP function. This study investigated the effects of two naturally occurring nonprotein thiols, i.e., glutathione (GSH) and N-acetylcysteine (NAC), on activation, function, and cellular–extracellular matrix interactions of the basement-membrane-degrading gelatinase, MMP-9. Our results reveal that NAC and GSH employ protein S-thiolation to inhibit organomercurial activation of pro-MMP-9. Gelatinase activity assays showed that GSH and NAC significantly inhibited MMP-9 but not MMP-2 function, implying isoform structural specificity. Immunoblot analyses, which suggested GSH interacts with MMP-9's active-site Zn, were corroborated by computational molecular modeling. Cell invasion assays revealed that NAC enhanced endostatin's ability to inhibit human cancer cell invasion. Collectively, these data demonstrate that nonprotein thiols suppress MMP-9 activation and function and introduce the prospect for their use in chemopreventive applications.

Section snippets

Oral squamous cell carcinoma cell lines

Five cell lines derived from HNSCCs of the tongue that developed in men between the ages of 25 and 70 years were obtained from the American Type Culture Collection. All of the SCC cell lines are aneuploid, are immortalized, have an epithelial morphology, and show growth rates ranging between 0.8 and 1.0 population doublings/day. Our laboratory has confirmed that these SCC cell lines retain many characteristics of oral mucosa, inclusive of preservation of antioxidant, cytoprotective enzymes and

Reduced, nonprotein thiols inhibit APMA-induced activation of pro-MMP-9

Gelatin zymography results demonstrated that inclusion of either 2.5 mM GSH or 2.5 mM NAC inhibited APMA-induced activation of pro-MMP-9 (Fig. 1A). In contrast, addition of 2.5 mM GSSG did not affect APMA-induced activation of MMP-9 (Fig. 1A). Equimolar levels of either GSH or NAC did not affect APMA-mediated MMP-9 activation. Further, although GSH and NAC did reduce APMA activation of pro-MMP-2, the extent of inhibition was appreciably less than the inhibitory effects noted with MMP-9 (data

Discussion

The oxidative stress that ensues during sustained inflammation perturbs the local redox balance, thereby establishing conditions that favor MMP activation [10]. Notably, increased MMP activity can promote carcinogenesis by numerous mechanisms that include enabling migration and invasion of cancer cells, release of sequestered growth factors, and facilitating tumor-associated angiogenesis. This study investigated the effects of two reduced nonprotein thiols, i.e.; GSH and NAC, on the activation

Acknowledgments

These studies were supported by NIH RO1 CA95901 and R21 CA111210 (S. R. Mallery).

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