Inhibitory effects of a benz[f]indole-4,9-dione analog on cancer cell metastasis mediated by the down-regulation of matrix metalloproteinase expression in human HT1080 fibrosarcoma cells

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Abstract

In our previous study, a synthetic benz[f]indole-4,9-dione analog, 2-amino-3-ethoxycarbonyl-N-methylbenz[f]indole-4,9-dione (SME-6), exhibited a potential anti-tumor activity. We, in this study, further explored the anti-metastatic and anti-invasive effect of SME-6 by determining the regulation of matrix metalloproteinases (MMPs). MMPs, zinc-dependent proteolytic enzymes, play a pivotal role in tumor metastasis by cleavage of extracellular matrix as well as non-matrix substrates. On this line, we examined the influence of SME-6 on the expressions of MMP-2, -9, membrane type 1-MMP (MT1-MMP), tissue inhibitor of metalloproteinases (TIMP-1, -2), and in vitro invasiveness of human fibrosarcoma cells. Dose-dependent suppressions of MMPs and TIMP-2 mRNA levels were observed in SME-6-treated HT1080 human fibrosarcoma cells detected by reverse transcriptase–polymerase chain reaction. TIMP-1 mRNA level, however, was induced in a dose-dependent manner. Gelatin zymographic analysis also exhibited a significant down-regulation of MMP-2 and -9 expression in HT1080 cells treated with SME-6 compared to controls. Furthermore, SME-6 inhibited the invasion, motility, and migration of tumor cells. Taken together, these data provide a possible role of SME-6 as a potential antitumor agent with the markedly inhibition of the metastatic and invasive capacity of malignant cells.

Introduction

Matrix metalloproteinases (MMPs) are a multigene family of zinc-dependent endopeptidases capable of degrading essentially all extracellular matrix components, and MMPs have been considered to play an important role in matrix degradation for tumor growth, invasion and tumor-induced angiogenesis (Westermarck and Kähäri, 1999). Among MMPs, MMP-2 and -9 are thought to play critical roles during tumor invasion and metastasis (Birkedal-Hansen et al., 1993, Itoh et al., 1998, Coussen et al., 2000).

Heterocyclic quinones with nitrogen atoms have been reported to exhibit antitumor and antibacterial activities (Silver and Holmes, 1968, Zee-Cheng and Cheng, 1982, Zee-Cheng et al., 1987). In our continuous efforts to develop novel antitumor agents we extended to synthesize benz[f]indole-4,9-dione analogs, heterocyclic pyrrole ring derivatives attached to 1,4-naphthoquinone based on the cytotoxic potential of 1,4-naphthoquinones (Fig. 1; Suh and Shin, 1992; SME-6). Benz[f]indole-4,9-dione analogs showed potential cell growth inhibitory activity against human cancer cells in our previous study. One probable mechanism of these compounds was suggested the catalytic inhibition of topoisomerase activity (Park et al., 2003). In addition, SME-6 induced G2/M cell cycle arrest and apoptosis in cultured human lung cancer cells (Lee et al., 2004). However, the exact mechanism of action of this compound remains still unclear.

In this study we further explore to investigate the influence of SME-6, a synthetic benz[f]indole-4,9-dione analogue, on the gene expression of MMP-2,-9, membrane type 1-MMP (MT1-MMP), tissue inhibitor of metalloproteinase-1,-2 (TIMP-1,-2), and in vitro invasiveness of human fibrosarcoma cells.

Section snippets

Materials

All media for cell culture were purchased from Gibco BRL (Grand Island, NY, USA). Human type I collagen, bovine serum albumin, and gelatin were purchased from Sigma (St. Louis, MO, USA). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA). All other chemicals used were of reagent grade. SME-6 was synthesized as described previously (Suh and Shin, 1992) and dissolved in dimethyl sulfoxide (DMSO) for use.

Cell culture

HT1080 human fibrosarcoma cells were grown in Dulbecco's modified Eagle's medium

Results

To investigate whether SME-6 can inhibit the expression of MMP-2 and -9, HT1080 human fibrosarcoma cells were treated with the compound for various concentrations, and MMP-2 and MMP-9 mRNA levels were determined by semi-quantitative RT-PCR technique. As shown in Fig. 2A, mRNA transcripts for MMP-2 and -9 were clearly inhibited in SME-6 treated cells. Down-regulation of MMP-2 and -9 expression by treatment of SME-6 was observed in a dose-dependent manner. To further explore the modulation of

Discussion

The aminoquinones are an interesting class of compounds with their remarkable anticancer activities (Skibo et al., 1994). Moreover, the planar tri- and tetracyclic quinones such as adriamycin have been found to act as intercalating agents (Marshall and Ralph, 1985). It is also known that benzo[f]indole-4,9-dione derivatives possess a variety of pharmacological activities including anticancer, virus-static, antibacterial, tuberculostatic, antianaphylastatic, fungicide, and anticoagulant effects (

Acknowledgments

This work was supported in part by a Korea Research Foundation Grant (KRF-2003-E00004).

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