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.

Fig. 1

Reduced nonprotein thiols inhibit activation of pro-MMP-9 by a mechanism that entails S-thiolation. (A) The effects of nonprotein thiols on APMA-mediated activation of pro-MMP-9 were determined by gelatin zymography. The enzyme-digested bands are observed as white bands against a dark background. Lane assignments: (1) pro-MMP-9, (2) pro-MMP-9 + 2.5 mM GSH, (3) pro-MMP-9 + 2.5 mM NAC, (4) pro-MMP-9 + 2.5 mM GSSG, (5) pro-MMP-9 + APMA, (6) pro-MMP-9 + 2.5 mM GSH + APMA, (7) proMMP-9 + 2.5 mM NAC + APMA, (8) pro-MMP-9 + 2.5 mM GSSG + APMA. (B) The nature of the reduced thiol pro-MMP-9 interaction was assessed by inclusion of the biotinylated GSH analogue, BioGee, followed by introduction of the disulfide-reducing agent, DTT (blot a). Western blot analyses were then conducted on the same washed membrane to confirm the integrity of the proMMP-9 protein (blot b). Lane assignments: (1) pro-MMP-9 only, (2) pro-MMP-9 + 100 μM BioGee, (3) pro-MMP-9 + 100 μM BioGee + 2 mM DTT, (4) proMMP-9 + 100 μM BioGee + 3 mM DTT, (5) pro-MMP-9 + 100 μM BioGee + 5 mM DTT, (6) pro-MMP-9 + 100 μM BioGee + 10 mM DTT. (C and D) Gelatin zymography was used to assess the effects of reduced thiols on release and subsequent activation of MMP-2 and MMP-9 which was secreted into the conditioned medium by cultured HNSCC cells as well as (E) cells isolated from AIDS-related Kaposi sarcoma (AIDS-KS). To enhance cellular release of MMPs, HNSCC and AIDS-KS cultures were serum deprived (SD) for 48 h, and conditioned medium was collected during this time. Lane assignments: (C) (1) HNSCC 4 48 h SD, (2) HNSCC 4 24 h SD + 24 h 25 mM NAC, (3) HNSCC 4 48 h SD + APMA, (4) HNSCC 4 24 h SD + 24 h 25 mM NAC + APMA, (5) MMP-2/MMP-9 standards; (D) (1) HSCC 9 24 h SD, (2) HNSCC 9 24 h SD + 24 h + 25 mM NAC, (3) HNSCC 9 48 h SD + APMA, (4) HNSCC 9 24 h SD + 24 h 25 mM NAC + APMA, (5) MMP-2/MMP-9 standards; (E) (1) KS 48 h SD, (2) KS 24 h SD + 25 mM NAC, (3) KS 48 h SD + APMA, (4) KS 24 h SD + 24 h 25 mM NAC + APMA, (5) MMP-2/MMP-9 standards.

Fig. 2

Reduced nonprotein thiols interact with MMP-9′s active-site Zn²⁺ molecule. (A) Immunoblot analyses were conducted using the biotinylated GSH analogue, BioGee, to determine the interaction of reduced nonprotein thiols with the active-site Zn molecule. (B) Western blot analyses were then conducted on the same washed membrane to confirm the integrity of the MMP-9 protein. Lane assignments were: (1) active MMP-9 with no incubation, (2) active MMP-9 + vehicle (ethanol) control with no incubation, (3) active MMP-9 + vehicle + 4 h incubation, (4) active MMP-9 + 464 μM (800×) BioGee, (5) active MMP-9 + 464 μM TPEN first, followed by 464 μM BioGee, (6) active MMP-9 + 464 μM BioGee first, followed by 464 μM TPEN, (7) active MMP-9 + 1160 μM (2000×) BioGee, (8) active MMP-9 + 1160 μM TPEN first, followed by 1160 μM BioGee, and (9) active MMP-9 + 1160 μM BioGee first, followed by 1160 μM TPEN.

Fig. 3

N-acetylcysteine augments endostatin's ability to suppress invasion of HNSCC cells. The ability of NAC and endostatin to inhibit the ability of calcein-loaded HNSCC cells to invade a synthetic basement membrane was evaluated using a commercially available cell invasion assay kit. Notably, in each cell line (n = 5) and in every experiment conducted (n = 8), the combination of NAC pretreatment with inclusion of NAC and endostatin during invasion inhibited HNSCC invasive capacities (p ≤ 0.05, Yates corrected χ² test).

Fig. 4

A model for the complex of active MMP-9 with N-acetylcysteine. The atomic coordinates used for the protein are from the structure of pro-MMP-9 (Accession No. 1L6J in the Protein Data Bank), with the prosequence 1–105 removed. The inhibitor is modeled into the groove on the surface of the protein that is exposed upon loss of the prosequence, in a position analogous to that of Cys 99, which in the structure is coordinated to the active-site zinc. N-acetylcysteine is proposed to coordinate in a similar fashion, as shown. The remaining ligands of the zinc are, clockwise from bottom left, His 411, His 405, and His 401 (positioned behind the zinc). We speculate that NAC (and also GSH) undergoes a slow binding reaction with the catalytic-site Zn²⁺, resulting in formation of a nonprotein thiol–Zn²⁺ complex.

Fig. 5

Docking of the MMP-9/NAC complex. (A) MMP-9 protein structure showing both zinc sites. The active-site zinc (on right, gray sphere), within the potential docking cleft, is coordinated by three histidines represented as sticks with the following atom coloring: carbon—green, nitrogen—blue, oxygen—red. Hydrogen atoms are not shown in the protein structure. The NAC molecule used in the docking study is shown above the MMP-9 structure. The NAC geometry was optimized at the B3LYP/6-31 + G* level of theory using Gaussian03 software [29]. The atom coloring is as above with the following additions: sulfur—yellow, hydrogen—white. (B) A docked structure of the MMP-9/NAC complex showing NAC positioned in the docking cleft nearest the active-site zinc, with the NAC sulfur within coordination distance of the metal. This structure was determined using the computational docking procedure described in the text. Hydrogen atoms are not shown. Atom coloring is as described above. (C) A model of the MMP-9/NAC complex in which the NAC ligand is coordinated to the active-site zinc analogous to Cys 99 in the crystal structure of pro-MMP-9 taken from the Protein Data Bank (Accession No. 1L6J) [45]. A partial geometry optimization was performed using the ONIOM method [46] as implemented in Gaussian03 [29] at the RHF/LANL2MB:UFF level of theory. The NAC ligand, zinc atom, and three coordinated histidines were included in the quantum mechanical region (RHF/LANL2MB), and the remaining portion of the MMP-9 protein was treated at the molecular mechanics level (UFF). Atom coloring is as described above. All graphical representations were generated with PyMol [47].