Cancer Letters

Cancer Letters

Volume 235, Issue 2, 28 April 2006, Pages 248-259
Cancer Letters

Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line

https://doi.org/10.1016/j.canlet.2005.04.033Get rights and content

Abstract

Anthocyanins, present in various fruits and vegetables as natural colorant, have been well characterized to be involved in various bioactive properties and are wildly used for their antioxidant properties. Furthermore, recent studies have revealed pleiotropic anticancer and antiproliferative capabilities of anthocyanin. Berry extract contains high amounts of anthocyanins and is commonly used in diet or in some therapeutic applications. In this study, we first observed that cyanidin 3-rutinoside and cyanidin 3-glucoside (extracted from Morus alba L.) exerted a dose-dependent inhibitory effect on the migration and invasion, of highly metastatic A549 human lung carcinoma cells in absence of cytotoxicity. The results showed that cyanidin 3-glucoside and cyanidin 3-rutinoside treatments could decrease the expressions of matrix matalloprotinase-2 (MMP-2) and urokinase-plasminogen activator (u-PA) in a dose-dependent manner and enhance the expression of tissue inhibitor of matrix matalloprotinase-2 (TIMP-2) and plasminogen activator inhibitor (PAI). Further analysis with semi-quantitative RT-PCR showed that these alterations were all on the transcriptional level. Further, a treatment of cyanidin 3-rutinoside and cyanidin 3-glucoside also resulted in an inhibition on the activation of c-Jun and NF-κB. Together, these result suggested that anthocyanins could decrease the in vitro invasiveness of cancer cells and therefore, may be of great value in developing a potential cancer therapy.

Introduction

Anthocyanins are a group of natural phenolic compounds responsible for the coloring of many plants, flowers, and fruits. In earlier studies, it has been shown that anthocyanin and other phenolic compounds may have potentially beneficial effects, including reducing the risk of cardiovascular diseases and cancers with antioxidant, anti-inflammatory, and chemoprotective properties [1], [2]. The exact related mechanisms were still uncertain, however, it may be contributed to their ability to suppress the proliferation [3], [4], [5] and angiogenesis [6], in addition to the induction of cancer cell apoptosis [7]. These compounds have also been reported to be able to scavenge reactive oxygen species, inhibit low-density lipoprotein oxidation in vitro, prevent platelet aggregation and decrease serum lipids in cholesterol-fed rabbits [8], [9], [10] while delphinidin could inhibit the invasiveness of human fibrosarcoma HT-1080 in vitro [11].

The progressing of a tumor from being in situ to invasive is a major prerequisite for cancer metastasis [12] and involves the acquisition of cell motility, surface adhesion properties, and activity of extracellular proteases. For invasion, a cancer cell requires increased migration, various cytophysiological changes including a loss of cell–cell adhesion along with a gain of cell–matrix adhesion, and increased expression and activation of extracellular proteases to degrade the extracellular matrix (ECM) and allow cell invasion and metastasis [12], [13], [14].

Extracellular proteases play an important role in immune functions, wound healing, fetus implantation, angiogenesis and cancer cell invasion [15]. Degradation of ECM by cancer cells via protease, such as serine proteinase, metalloproteinases (MMPs), cathepsins, may lead to the separation of intercellular matrix to promote the mobility of cancer cells and eventually result in metastasis [15]. Of these proteases, serine proteinase and MMPs are the most vital ones for degradation of basement membrane and therefore involved in cancer migration and invasion [16]. The MMPs, a group of zinc-dependent ECM-degrading enzymes for the proteolysis of ECM proteins such as collagen, proteoglycan, fibronectin, elastin and laminin [17], are thought to be critical in tumor invasion, metastasis and angiogenesis [12]. Both MMP-2 and MMP-9 are abundantly expressed in various malignant tumors [18] and contribute to invasion and metastasis as documented in many reports [19], [20], [21]. Furthermore, a serine proteinase, urokinase-type plasminogen activator (u-PA), may promote a proteolytic cascade by converting plasminogen to its active form, plasmin, which in turn may cleave and activate MMPs to enhance tissue remodeling by degrading ECM components [22]. Meanwhile, the activities of MMPs and u-PA are prone to the inhibition of endogenous tissue inhibitor of metalloproteinases (TIMPs) and plasminogen activator inhibitor (PAI), which are specific inhibitors of MMPs and PAI, respectively, and the imbalance between MMPs and TIMPs may contribute to degradation or deposition of ECM [23].

However, these studies on functions of anthocyanins have been mainly focused on the effects of anti-oxidation or suppression of tumor cell proliferation, whereas the effect of anthocyanins on migration and invasion of tumor cells has not been clearly clarified. Since cancer metastasis and invasion are highly related to degradation of extracellular matrix, intercellular adhesion and cellular motility, in this study, by looking at the impact of cyanidin 3-glucoside and cyanidin 3-rutinoside on several relevant proteases, including MMP-2, u-PA, PAI and TIMP-2, of A549, a human lung cancer cells with a high metastasis capability, the effect of cyanidin 3-glucoside and cyanidin 3-rutinoside on the invasion, motility, adhesion and related signaling pathways was investigated.

Section snippets

Extraction and purification of anthocyanins from mulberry

Weighted fruits of mulberry (Morus alba L.; purchased from a private berry farm in Taichung county, Taiwan) were extracted by an incubation with 0.1% HCl in methanol for over 48 h in the dark. The slurry and remaining solvent was removed by filtration and evaporation at 30 °C using a rotary evaporator, respectively. The obtained pigments were then evenly dissolved in deionized water and this concentrated solution was chromatographed via a 40 cm×2.5 cm Diaion HP-20 column and eluted with methanol.

Isolation of cyanidin 3-rutinoside and cyanidin 3-glucoside from mulberry

Anthocyanin components were extracted from mulberry (Morus alba L.) and after a separation by Diaion HP-20, an anthocyanins fraction of 2.5% was recovered. The purity of the collected compounds after a separation by Bio-Gel P-2 column was estimated to be 99% HPLC and LC-MS. The compounds were identified to be cyanidin 3-rutinoside (peak I from Bio-Gel P-2 [M+H]+, m/z 595) and cyanidin 3-glucoside (peak II from Bio-Gel P-2 [M+H]+, m/z 449) by LC-MS. These were also confirmed by the spiking with

Discussion

Many studies have demonstrated that anthocyanins, natural pigments from various plants, flowers, and fruits, have inhibitory actions against cell proliferation and were able to reduce deleterious effects of reactive oxygen species [32], and therefore, were regarded as chemopreventive agents. Recently, anti-invasion agents have been defined as a new class of cancer chemopreventive agent [33]. The finding of the present study showed that cyanidin 3-rutinoside and cyanidin 3-glucoside have strong

Acknowledgements

This study was supported in part by National Science Council, Republic of China (NSC 93-2320-B-040-061).

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