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

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. KeywordsGlutathione; Nonprotein thiols; MMPs; Cell invasion; Thiolation; Chemoprevention; Free radicals Both physiological processes such as angiogenesis and pathologic events like tumor cell invasion are dependent on proteolytic degradation of the extracellular matrix (ECM) by enzymes that include matrix metalloproteinases (MMPs) [1]. In physiological states, MMP activity is closely regulated at three distinct levels, i.e., gene expression, enzyme activation, and enzyme inhibition [2,3]. Conversely, it has been proposed that loss of MMP regulation, by prolonged MMP activation and/or concurrent inactivation of tissue inhibitors of MMPs (TIMPs), contributes to diseases, including cancer [2,3]. Consistent with their putative carcinogenesis-facilitating roles, increased MMP activity has been detected in a variety of human cancers, including colorectal, breast, lung, prostate, and head and neck squamous cell carcinoma (HNSCC) [4][5][6][7][8]. Studies conducted on HNSCC resected tumors revealed that MMP functional increases were apparent in tumor as well as in inflammatory and stromal cells, implying a co-inductive effect on tumor and stromal MMPs [6,7]. Furthermore, these localized increases in MMP activity can promote carcinogenesis by several mechanisms that include enabling tumor cell invasion of basement membranes and release of proangiogenic growth factors formerly sequestered in the ECM. Results from clinical studies, which show a positive correlation between the e...

Formulation and Characterization of Injectable Poly(dl-lactide-co-glycolide) Implants Loaded with N-Acetylcysteine, a MMP Inhibitor

“…In addition to cytoprotection, these antioxidant properties contribute to NAC's ability to inhibit proMMP activation and suppress mature enzyme function. Our results, which demonstrate that only the reduced, thiol containing forms of glutathione and NAC inhibit proMMP activation and suppress MMP-9 activity (14), emphasize the need for polymer microclimate regulation in order to preserve thiol integrity.…”

“…The ampoules were placed in an incubator maintained at 37°C and shaken at 100 RPM. At predetermined time intervals (1,3,7,14,21, and 28 days), three ampoules were taken out, broken, and release medium was suitably diluted and cumulative amount of released NAC was determined by HPLC.…”

“…MMP-9, which degrades collagen type IV, is a key enzyme involved with basement membrane degradation and subsequent progression of precancerous oral epithelial lesions to overt cancer (11,13). Our labs have recently shown that as a result of its reduced thiol, NAC: (1) inhibits activation of the proform of MMP-9 (an MMP that is involved in degradation of collagen type IV-a key component of the basement membrane), and (2) serves as a surrogate propeptide and inhibits function of the previously activated MMP-9, putatively by blocking the enzyme's active site (14). The reduced nonprotein thiol-MMP interactions have functional consequences as shown by inhibition of invasion of cultured HNSCC cells following introduction of NAC (14).…”

“…Our labs have recently shown that as a result of its reduced thiol, NAC: (1) inhibits activation of the proform of MMP-9 (an MMP that is involved in degradation of collagen type IV-a key component of the basement membrane), and (2) serves as a surrogate propeptide and inhibits function of the previously activated MMP-9, putatively by blocking the enzyme's active site (14). The reduced nonprotein thiol-MMP interactions have functional consequences as shown by inhibition of invasion of cultured HNSCC cells following introduction of NAC (14). Due to the importance of extracellular matrix degradation in tumor associated angiogenesis and tumor cell invasion, PLGA injectable implants that provide sustained therapeutic levels of demonstrated MMP inhibitors, NAC for example, have the potential to markedly inhibit locoregional HNSCC tumor recurrence and deter the formation of second HNSCC primary tumors.…”

Diverse roles of matrix metalloproteinases and tissue inhibitors of metalloproteinases in neuroinflammation and cerebral ischemia

“…1) (Gasche et al, 2006). Protease-independent activation of the MMPs by S-nitrosylation or oxidation can unmask the catalytic domain producing activation of MMPs without pro-domain cleavage (Gu et al, 2002;Meli et al, 2003;Pei et al, 2006).…”