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Inhibitory effect of N-acetylcysteine on invasion and MMP-9 production of T24 human bladder cancer cells.

Kawakami S, Kageyama Y, Fujii Y, Kihara K, Oshima H.

Department of Urology and Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-Ku, Tokyo 113-8519, Japan. s-kawakami@med.tmd.ac.jp

BACKGROUND: MMPs play a crucial role in the process of cancer invasion and metastasis.

METHODS: The influence of NAC on invasion and MMP-9 production of human bladder cancer cell line T24 was investigated using an in vitro invasion assay, gelatin zymography, Western and Northern blot analyses and RT-PCR assays.

RESULTS: TPA increased the number of invading T24 cells through reconstituted basement membrane more than 10-fold compared to basal condition. NAC inhibited TPA-enhanced invasion dose-dependently. TPA increased the MMP-9 production by T24 cells without altering expression of TIMP-1 gene, while NAC suppressed TPA-enhanced production of MMP-9. Neither TPA nor NAC altered TIMP-1 mRNA level in T24 cells. In vitro experiments demonstrated that MMP-9 was directly inhibited by NAC but was not influenced by TPA.

CONCLUSION: NAC limits invasion of T24 human bladder cancer cells by inhibiting the MMP-9 production in addition to a direct inhibition of MMP-9 activity.

PMID: 11299737 [PubMed - indexed for MEDLINE]

We have recently demonstrated that interleukin-1 beta (IL-1 beta) stimulates matrix metalloproteinase-9 (MMP-9) induction.

In this study we have investigated the roles of superoxide and extracellular signal-regulated kinase (ERK) activation in MMP-9 induction following exposure to IL-1 beta. IL-1 beta stimulated biphasic ERK activation in vascular smooth muscle (VSM) cells, a transient activation that reached a maximum at 15 min and declined to baseline levels within 1 h, and a second phase of sustained ERK activation lasting up to 8 h.

To determine the role of ERK in IL-1 beta-stimulated MMP-9 induction, we treated cells with the specific ERK pathway inhibitor PD-98059 at different time intervals after IL-1 beta stimulation. Addition of PD-98059 up to 4 h after IL-1 beta stimulation significantly inhibited MMP-9 induction, suggesting a role for sustained ERK activation in MMP-9 induction.

IL-1 beta treatment stimulated superoxide production in VSM cells that was inhibited by pretreatment of cells with the superoxide scavenger N-acetyl-L-cysteine (NAC) and also by overexpression of the human manganese superoxide dismutase (MnSOD) gene.

Treatment of VSM cells with NAC selectively inhibited the sustained phase of ERK activation without influencing the transient phase, suggesting a role for reactive oxygen species in sustained ERK activation.

In addition, both NAC treatment and MnSOD overexpression significantly inhibited IL-1 beta-stimulated MMP-9 induction (P < 0.05).

The results demonstrate that IL-1 beta-dependent MMP-9 induction is mediated by superoxide-stimulated ERK activation.

PMID: 11709424 [PubMed - indexed for MEDLINE]

Increased activity of matrix metalloproteinase-2 in human glial and neuronal cell lines treated with HIV-1 gp41 peptides.

Part of the neurodegenerative cascade in AIDS dementia may involve overexpression of matrix metalloproteinases (MMPs).

Here, we examined the possible effect of HIV-1 gp41, which has been shown as a key determinant associated with pathogenesis of AIDS dementia, on the activity of MMPs using human neuronal and glial cell lines.

Zymographic analysis revealed that treatment with the gp41 peptide (aa 583-599) for 24 h markedly elevated the activity of MMP with Mr 66 kDa in the cultured media of glioblastoma cell line T98G in a concentration-dependent manner as well as of neuroblastoma cell line SK-N-SH despite of lower magnitude of the activity.

In contrast, the immediately adjacent gp41 peptide (aa 598-613) as well as the reverse peptide (aa 598-583) had a little effect.

Recombinant gp41 protein containing extracellular domain also elicited a similar effect, although with a lesser extent.

This 66 kDa MMP was confirmed as gelatinase A (MMP-2) based on the results of its activity dependent on Ca2+ and inhibited in the presence of 1,10-phenanthroline or EDTA, as well as its specific immunoreactivity on the Western blot.

N-acetyl cysteine (NAC) downregulated this gp41 peptide-induced MMP-2 activity in T98G.

The soluble form of amyloid precursor protein (sAPP), which is synthesized in the Escherichia coli system, also inhibited the MMP-2 activity in vitro.

Taken together, these results implicate that high production of HIV-1 gp41 or its metabolites containing aa 583-599 within central nervous system (CNS) could result in the increased activity of MMP-2 and that the extracellular deficiency of reducing agent or decreased level of sAPP within CNS could exacerbate this gp41-induced MMP-2 activity.

PMID: 9699154 [PubMed - indexed for MEDLINE]

• Circulation. 1998 Jun 23;97(24):2382-3.

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N-acetyl-cysteine decreases the matrix-degrading capacity of macrophage-derived foam cells: new target for antioxidant therapy?

Galis ZS, Asanuma K, Godin D, Meng X.

Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA. zgalis@emory.edu

BACKGROUND: Atherosclerotic plaque destabilization triggers clinical cardiovascular disease and thus represents an attractive therapeutic target.

Weakening of tissue through the action of matrix-degrading enzymes, called matrix metalloproteinases (MMPs), released by resident macrophages was previously implicated in unstable vascular syndromes.

METHODS AND RESULTS: We used a hypercholesterolemic rabbit model of atherosclerosis to investigate the gelatinolytic activity associated with macrophage-derived foam cells (FCs). Gelatinolytic activity and expression of MMP-9 but not of MMP-2 cosegregated with macrophage FCs in aortic lesions. Macrophage-derived gelatinases were further investigated in vitro.

MMP-9 was identified as the main macrophage-derived gelatinase in cells isolated from aortic lesions and from granuloma induced in the same rabbits to increase cell yield. Importantly, detection of activated MMP-9 in the FC culture medium supports the notion that these cells can independently initiate processing of secreted MMP zymogens to active enzymes. We further examined whether FC gelatinolytic activity is dependent on the presence of reactive oxygen species (ROS).

We found that treatment (1 to 5 days) with 1 to 10 mmol/L N-acetyl-L-cysteine (NAC), an ROS scavenger, decreased not only gelatinolytic activity but also gelatinase expression by FCs.

Similarly, NAC treatment of explanted lesions abolished in situ gelatinolytic activity and MMP-9 expression.

CONCLUSIONS: Macrophage FCs are an abundant source of gelatinolytic activity that can be inhibited in vitro and in situ by NAC. This newly described action of antioxidant therapy might prove useful to inhibit matrix degradation and to improve vascular stability.

PMID: 9641697 [PubMed - indexed for MEDLINE]

 
Alveolar macrophage-mediated elastolysis: roles of matrix metalloproteinases, cysteine, and serine proteases.

Chronic obstructive pulmonary disease (COPD) is a common lung disease with cigarette smoking as the major etiological factor, but only 15% of smokers develop COPD.

Destruction of lung elastin observed in COPD is mediated by many enzymes, including cysteine, serine, and matrix metalloproteinases (MMP). The contribution of these enzymes to the lung elastolytic load, released from alveolar macrophages collected from nonsmokers, healthy smokers, and COPD patients, was examined by radiolabeled elastin as substrate in the presence of specific enzyme inhibitors.

The activity of MMP was further examined by zymography and Western blotting. COPD macrophages degraded more elastin than either of the other groups. Elastolysis was greatest in the initial 24 h. Through the 72-h culture period, the contribution to elastolysis of serine elastases decreased, MMP increased, and cysteine elastases remained constant.

The increased release of elastolytic enzymes in COPD subjects may explain why some smokers develop COPD. This difference may be due to unknown susceptibility factors. Serine proteases play a significant role; however, other enzymes, particularly the MMP, deserve further investigation.

PMID: 12225964 [PubMed - indexed for MEDLINE]

Angioprevention': angiogenesis is a common and key target for cancer chemopreventive agents.

Tosetti F, Ferrari N, De Flora S, Albini A.

Molecular Biology Laboratory, National Cancer Research Institute (IST), Genova, Italy.

The potential to block tumor growth by inhibition of the neoangiogenic process represents an intriguing approach to the treatment of solid tumors.

The high proliferation rate in the tumor deprived of proper vascularization would be balanced by cell death due to lack of diffusion of nutrients and oxygen.

Matrix metalloproteinases (MMPs), angiogenic growth factors, and their receptors are the main targets of an increasing number of clinical trials approved to test the tolerance and therapeutic efficacy of antiangiogenic agents.

We observed that a series of substances proposed as possible cancer chemopreventive agents show antiangiogenic properties when tested in in vitro and in vivo angiogenesis models.

We demonstrated that N-acetyl-l-cysteine is able to reduce the invasive and metastatic potential of melanoma cells, and to inhibit endothelial cell invasion by direct inhibition of MMP activity.

We also showed that epigallocatechin gallate (EGCG), a flavonoid from green tea that possesses chemopreventive activity in experimental and epidemiological studies, is a potent inhibitor of MMP-2 and MMP-9.

Angiogenesis has also been demonstrated to be a target for nonsteroidal anti-inflammatory drug chemopreventive activity. Based on these data, we hypothesize that other chemopreventive agents, including natural or synthetic retinoids, steroid hormone antagonists, peroxisome proliferator-activated receptor gamma ligands, vitamin D, and protease inhibitors, might have antiangiogenesis as an important mechanism of action, a novel concept we will term 'angioprevention'.

We analyze the mechanisms on how and why chemopreventive agents could exert antiangiogenic effects aimed at controlling tumor growth, and their potential use in the clinic.

Publication Types:

• Review

• Review, Tutorial

PMID: 11772931 [PubMed - indexed for MEDLINE

MMP-28, a new human matrix metalloproteinase with an unusual cysteine-switch sequence is widely expressed in tumors.

We report the discovery, cloning, and characterization of a novel human matrix metalloproteinase (MMP-28) cDNA gene.

The deduced 520-amino-acid sequence of MMP-28 includes a signal peptide, a prodomain with an unusual cysteine-switch PRCGVTD motif followed by the furin cleavage RRKKR site, a catalytic domain, a hinge-region and a hemopexin-like domain.

On the basis of their structural characteristics, MMP-28 belongs to the MMP-19 subfamily. The genomic MMP-28 gene uniquely mapped to chromosome 17q11.2 includes eight exons and seven introns. The broad range of expression in carcinomas as well as normal adult and fetal tissues suggests an important functional role for MMP-28.

PMID: 11255011 [PubMed - indexed for MEDLINE]