The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in progression of atherosclerotic lesions. The role and importance of the signaling pathway in the transcriptional regulation of MMP-9 in human aortic smooth muscle cells (HASMC) was examined. Tumor necrosis factor-alpha (TNF-alpha) stimulated the secretion of MMP-9 in HASMC, as shown by zymography and immunoblot analysis. At the transcriptional levels, TNF-alpha also stimulated the 5'-flanking 710-bp promoter activity of MMP-9. Transcription factors NF-kappaB binding site (-601) and AP-1 binding site (-82) were identified as the cis-elements for TNF-alpha activation, as determined by gel shift assay and mutation analysis. Treatment with U0126, an inhibitor of the extracellular signal-regulated kinase (ERK), significantly downregulated TNF-alpha-induced MMP-9 expression and promoter activity, whereas the inactive analog U0124 had no effect. Furthermore, the transactivation of TNF-alpha-stimulated NF-kappaB and AP-1 was inhibited by U0126 treatment. Finally, the transient transfection of HASMC with dominant negative Ras (RasN17) suppressed TNF-alpha-induced ERK activity, MMP-9 production, and promoter activity. Overexpression of RasN17 also abolished the TNF-alpha-stimulated NF-kappaB and AP-1 activity. In conclusion, the findings herein indicate the activation of the Ras/ERK pathway contributes to the induction of MMP-9 expression in HASMC. In addition, the transcription factors NF-kappaB and AP-1 that are involved in the Ras/ERK-mediated control of MMP-9 regulation on HASMC in response to TNF-alpha have now been identified.

Our previous studies have clearly shown that the angiogenic enzymes, matrix metalloproteinase (MMP) -2/9, are directly involved in human hepatic tumorigenesis and metastasis and suggest that the MMP-2/9 inhibitors, which have dual inhibitory activities on enzyme activity and transcription, represent the best candidates for achieving tumor regression. Many anti-cancer drugs have strong cellular cytotoxicity and side effects, indicating that strong anti-cancer drugs that have no or minimal cytotoxicity and side effects need to be developed. The specific aim of the present study was to develop powerful anti-cancer drugs with specific tumor regression and anti-metastatic potential having the dual inhibitory activities of specific MMP-2 and -9 enzyme activities and gene transcription at the molecular level. Caffeic acid (CA), a strong and selective MMP-9 activity and transcription inhibitor, was isolated from the plant Euonymus alatus and its derivative, caffeic acid phenethyl ester (CAPE), was synthesized. CA and CAPE selectively inhibited MMP-2 and -9 but not -1, -3, -7, or cathepsin K. Treatment of HepG2 cells with CA (100 microg/mL) and CAPE (5 microg/mL) suppressed phorbol 12-myristate 13-acetate (PMA) -induced MMP-9 expression by inhibiting the function of NF-kappaB, but not AP-1. We confirmed that CA and CAPE suppressed the growth of HepG2 tumor xenografts in nude mice in vivo. The subcutaneous and oral administrations of CA and CAPE significantly reduced the liver metastasis. These results confirm the therapeutic potential of the compounds and suggest that the anti-metastatic and anti-tumor effects of CA and CAPE are mediated through the selective suppression of MMP-9 enzyme activity and transcriptional down-regulation by the dual inhibition of NF-kappaB as well as MMP-9 catalytic activity.

Hepatitis B virus (HBV) X protein (HBx) has been shown to be essential for the development of hepatocellular carcinoma (HCC). Recently, we have found that HBx causes the progression of liver cancer through down-expression of PTEN, known as a tumor suppressor gene (1). The prognosis for HCC depends mainly on the clinicopathological characteristic regarding invasion and metastasis. The expression of matrix metalloproteinase (MMP)-9 has been implicated as playing an important role in HCC invasion and metastasis. We previously reported that HBV infection increased the invasiveness of hepatocytes and HCC cells through the transcriptional activation of MMP-9 (2). The HBx was shown to activate the mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI-3K) signal cascade, which is essential for activation of transcription factors such as activating protein (AP)-1 and nuclear factor (NF)-kappaB. In this study, we show that the HBx protein stimulates the activities of the PI-3K-Akt/ protein kinase B (PKB) as well as extracellular signal-regulated kinase 1/2 (ERK 1/2) in HBx-transfected cells. Furthermore, we have shown that enhanced expression of MMP-9 in HBx-transfected cells mediated by not only activation of AP-1 transcriptional activity through ERKs pathway but also activation of NF-kappaB transcriptional activity through PI-3K-AKT/PKB pathway, and was associated with the invasive potential. However, treatment with U0126 (known as the ERKs inhibitor) or wortmannin (known as the PI-3K inhibitor), but not SB203580 (known as the p38 MAPK inhibitor), markedly inhibited the expression of MMP-9 induced by HBx in HBx-transfected cells. Seemingly, the invasiveness of HBx-transfected cells was decreased by treating with U0126 or wortmannin, but not SB203580. These results clearly suggest that the HBx contributed to the transcriptional regulation of MMP-9 through the ERKs and PI-3K-AKT/PKB pathway, and increased an invasive potential of cells.

The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. Here we examined the effect of ascochlorin, a prenyl-phenol anti-tumor compound from the fungus Ascochyta viciae, on the regulation of signaling pathways that control MMP-9 expression in human renal carcinoma (Caki-1) cells. Ascochlorin reduced the invasive activity of Caki-1 cells and inhibited phorbol 12-myristate 13-acetate-induced increases in MMP-9 expression and activity in a dose-dependent manner. Reporter gene, electrophoretic mobility shift, kinase inhibitor assays, and in vitro kinase assay showed that ascochlorin inhibits MMP-9 gene expression by suppressing activation of the nuclear transcription factor activator protein-1 (AP-1) via the extracellular signalregulated kinase 1 and 2 pathway. The AP-1 family member most specifically affected by ascochlorin was Fra-1. Ascochlorin did not affect the activation of the c-Jun N-terminal or p38 kinase pathways. Moreover, transfection of Caki-1 cells with AP-1 decoy oligodeoxynucleotides resulted in the suppression of phorbol 12-myristate 13-acetate-induced MMP-9 expression and invasion. In conclusion, ascochlorin represents a unique natural anti-tumor compound that specifically inhibits MMP-9 activity through suppression of AP-1-dependent induction of MMP-9 gene expression.Ascochlorin (see Fig. 1A), a prenyl-phenol compound isolated from the fungus, Ascochyta viciae, was originally found to have antiviral antibiotic activity (1). In addition to its antiviral and anti-fungal activity, natural and synthetic derivatives of ascochlorin reduce serum cholesterol and triglyceride levels suppress hypertension and tumor development and ameliorate type I and II diabetes (2-9). Moreover, several ascochlorin derivatives have been reported to be potent agonists of nuclear hormone receptors, including peroxisome proliferator-activated receptor ␥, suggesting that the structure of ascochlorin would be useful in designing modulators of nuclear receptors (6, 10, 11, 13).The most well known extracellular matrix (ECM) 1 -degrading enzymes are the matrix metalloproteinases (MMPs). MMPs are a family of zinc-dependent endoproteinases that are capable of degrading all the components of the ECM. MMPs are structurally and functionally homologous proteins, with at least 20 known members. MMPs can be divided into four families based on structure and substrate specificity: collagenases, gelatinases, stromelysins, and membrane-associated MMPs. Among the previously reported human MMPs, gelatinase-A (MMP-2) and gelatinase-B (MMP-9) are key enzymes that degrade type IV collagen (14, 15). These two gelatinases share structural and catalytic similarities, but transcription of the MMP-2 and MMP-9 genes is independently regulated due to distinct arrays of regulatory elements in the gene promoters. Regulated expression of MMP-9 has been implicated in renal development, macrophage differentiation, atherosclerosis, inflammation, rheumatoid arthritis, and tumor invasion (16 -17). I...