Elsevier

Biomedicine & Pharmacotherapy

Volume 62, Issue 6, July–August 2008, Pages 366-372
Biomedicine & Pharmacotherapy

Original article
Resveratrol inhibits tumor necrosis factor-α-mediated matrix metalloproteinase-9 expression and invasion of human hepatocellular carcinoma cells

https://doi.org/10.1016/j.biopha.2007.09.006Get rights and content

Abstract

Resveratrol is an active polyphenol found in red wine that has anti-cancer effects, but the molecular mechanisms of resveratrol on tumor invasion inhibition have not been well documented. The aim of this study was to elucidate the effects of resveratrol on invasion ability of human hepatocellular carcinoma cells and TNF-α-mediated MMP-9 expression. The expression activity of MMP-9 was measured by zymography, RT-PCR and western blot analysis. The expression of NF-kappa B was measured by EMSA and western blot analysis. TNF-α induced the MMP-9 expression in HepG2 cells. Resveratrol significantly inhibited TNF-α-mediated MMP-9 expression in HepG2 cells. NF-kappa B inhibitor induced a marked reduction in MMP-9 expression, and it suggested that NF-kappa B could play an important role in TNF-α-mediated MMP-9 expression. Furthermore, resveratrol significantly suppressed TNF-α-mediated NF-kappa B expression and invasion of HepG2 cells. Our results showed that resveratrol inhibited TNF-α-mediated MMP-9 expression and invasion of human hepatocellular carcinoma cells. The inhibitory effects are partly associated with the downregulation of the NF-kappa B signaling pathway.

Introduction

Hepatocellular carcinoma (HCC) was the sixth most common cancer and the third most common cause of death from cancer [1], [2]. Invasion is a characteristic feature of HCC, which frequently shows early invasion into blood vessels often happened in early stage of HCC and intrahepatic metastasis and extrahepatic metastasis may occur subsequently [3]. Invasion and metastasis are fundamental properties of malignant cancer cells and the main factors related to the poor prognosis. The main physiologic barriers to the movement of cell into tissues is invasion of malignant cancer cells into the extracellular matrix (ECM) and basement membrane (BM), so the degradation of ECM and BM is one of the most important steps in the process of cancer invasion and metastasis [4]. Matrix metalloproteinases (MMPs) is a family of enzymes capable of degrading the constituents of ECM and BM, which is a crucial step involved in various stages of tumor progression, including tumor angiogenesis, tumor growth, and also local invasion and subsequent distant metastasis [5]. High MMP levels are associated with poor prognosis in cancer patients [6]. Matrix metalloproteinase-9 (MMP-9) is an endopeptidase of the large MMPs family [7]. An enhanced expression of MMP-9 has been shown to be associated with the progression and invasion of tumors [8], [9], [10]. Overexpression of MMP-9 is associated with the capsular infiltration of HCC and growth of small HCC has been reported [11], [12]. So inhibiting MMP-9 expression is necessary for HCC therapy.

Nuclear factor-kappa B (NF-kappa B) is a transcription factor discovered by Sen and Baltimore in 1986, in the kappa light chain of immunoglobulins in B cells [13]. NF-kappa B has been found an inducible and ubiquitously expressed transcription factor for genes involved in cell survival, cell adhesion, inflammation, differentiation, and growth [14]. Amplification and overexpression of NF-kappa B have been found in cervical, ovarian, gastric and breast carcinomas [15], [16], [17], [18]. A recent study demonstrated that early activation of NF-kappa B contributes probably to the acquisition of a transformed phenotype during hepatocarcinogenesis, whatever the etiology [19]. TNF-α is an elicitor of NF-kappa B activation [20]. Overexpression of NF-kappa B is also able to up-regulate the expression of MMP-9 and to enhance the invasive capacity of tumor cells [21], [22]. These observations suggested that, in hepatocellular carcinoma cells, the NF-kappa B signaling pathway might be critical for metastatic cell spread.

Resveratrol (3,5,4′-trihydroxystilbene), an active polyphenol found in red wine that is associated with many health benefits, most notably the mitigation of age-related diseases, including neurodegeneration, carcinogenesis and atherosclerosis [23], [24], [25]. The chemotherapeutic role of resveratrol could act at several stages of the multistep malignancy process [26]. However, the anti-cancer molecular mechanism of resveratrol was not elucidated fully. The inhibitory effects of resveratrol on the TNF-α-mediated expression of MMP-9 have not been further studied. In this study, we investigated whether resveratrol could affect MMP-9 expression and suppress the TNF-α-mediated expression of NF-kappa B signaling pathway during hepatocellular carcinoma cell invasion.

Section snippets

Reagents and chemicals

Resveratrol, gelatin, pyrrolidine dithiocarbamate (PDTC) and TNF-α were purchased from Sigma. Dulbecco's modified Eagle's medium (DMEM), trypsin, Trizol reagent and fetal bovine serum (FBS) were from Gibicol. ExScript™ RT reagent kit and Taq DNA polymerase were from Invitrogen. Goat monoclonal anti-human MMP-9 antibody was from NeoMarker. Rabbit polyclone anti-human NF-kappa B antibody was from Santa Cruz. Millicell invasion chambers were obtained from Millipore. RIPA buffer was purchased from

Effect of TNF-α-mediated MMP-9 activation in HepG2 cells

We used a specific kinase inhibitor, PDTC to examine the important role of NF-kappa B signaling pathway in the regulation of MMP-9 expression. We found the NF-kappa B-specific inhibitor PDTC at 100 μM could significantly suppress TNF-α-mediated MMP-9 activity with zymography assay (p < 0.05) (Fig. 1a,b). At the same time, MMP-9 mRNA expression was obviously inhibited by PDTC (p < 0.05) (Fig. 1c,d). It suggested that the expression of MMP-9 is partly regulated by the NF-kappa B signaling pathway.

Discussion

MMPs play a major role in promoting angiogenesis and tumor metastasis [27]. An enhanced expression of MMP-9 has been shown to be associated with the progression and invasion of tumors [28], [29]. NF-kappa B signaling pathway has been shown to correlate with MMP-9 expression and tumor invasion [30]. At the same time MMP-9 promoter consists NF-kappa B-binding sites, it was plausible to investigate the important roles of NF-kappa B signaling pathways in TNF-α-mediated MMP-9 activation. Previous

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