Carboxymethylations of chitosan and chitin inhibit MMP expression and ROS scavenging in human fibrosarcoma cells Chang-Suk Kong a , Jung-Ae Kim b , Byulnim Ahn b , Hee-Guk Byun c , Se-Kwon Kim a,b, * a Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Republic of Korea b Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea c Faculty of Science and Biotechnology, Kangnung National University, Gangneung 210-702, Republic of Korea 1. Introduction Oxidative stress is generally used to describe the steady-state level of oxidative damage in cells, tissues, or organs. It is caused by the reactive oxygen species (ROS), which are known to be a major factor in biological damage [1]. ROS include hydrogen peroxide and other chemical forms such as superoxide anion, hydroxyl radical and peroxyl radical. ROS are generally unstable and can highly active one or more unpaired electrons in the body and endogenously produced ROS are essential to life, being involved in several biological functions [2]. However, overproduction of ROS can induce significant damage to cell structures such as lipids, proteins and DNA [3]. The uncontrolled oxidative stresses due to excessive production of active oxygen or imbalance in body’s redox potential are associated with diverse pathological processes, leading to various cellular damages and diseases [4,5]. Further- more, ROS are involved in the progression of tumor-induced angiogenesis because several different matrix metalloproteinases (MMPs) can positively be regulated by the intracellular redox state [6]. MMPs play a substantial role in pathological process of major chronic diseases such as arthritis, inflammation, cardiovascular diseases and cancer [7]. Especially, MMP-2 (gelatinase-A, 72 kDa) and MMP-9 (gelatinase-B, 92 kDa) degrade components of the basement membrane and are responsible for tumor invasion and metastasis [8,9]. Overexpression of MMP-2 and MMP-9 in human fibrosarcoma cells induced markedly enhanced metastasis [10]. Therefore, to find MMP inhibitors would be valuable as a new class of cancer therapeutic targets of current interest. Chitin is a naturally abundant mucopolysaccharide found in the shell of crustaceans, the cuticle of insects and the cell wall of some fungi and microorganisms. Chitosan is derived from chitin by an alkaline deacetylation. Chitosan and chitin consist of 2-amino-2- deoxy-(1-4)-b-d-glucopyranose residues (d-glucosamine units) and 2-acetamido-2-deoxy-(1-4)-b-d-glucopyranose residues (N- acetyl-d-glucosamine units), respectively [11]. Chitosan and chitin have been used considerably for its commercial applications in biomedical, food and chemical industries [12]. However, their biological activities were found only in acidic condition due to their major disadvantages such as low solubility, low absorbability or non-digestible, high molecular weight and high viscosity. Thereby, the studies on the chemical modification or application of chitosan and chitin have been conducted to develop water-soluble derivatives of chitosan and chitin [13,14]. Carboxymethylation and deacetylation reactions were occupied in converting them to Process Biochemistry 45 (2010) 179–186 ARTICLE INFO Article history: Received 4 March 2009 Received in revised form 31 August 2009 Accepted 3 September 2009 Keywords: Carboxymethyl-chitosan Carboxymethyl-chitin ROS MMP TIMP NF-kB ABSTRACT The water-soluble derivatives of chitosan and chitin, carboxymethyl-chitosan (CM-chitosan) and carboxymethyl-chitin (CM-chitin), were synthesised by means of carboxymethylation reaction. Their antioxidative and matrix metalloproteinase-2 and -9 (MMP-2 and -9) inhibitory effects were investigated in HT1080 human fibrosarcoma cells. Treatment with CM-chitosan and CM-chitin suppressed the formation of intracellular reactive oxygen species (ROS), protein oxidation and lipid peroxidation in a concentration-dependent manner. In addition, a protective effect against oxidative damage of purified genomic DNA was observed in the presence of CM-chitosan and CM-chitin. Moreover, CM-chitosan and CM-chitin reduced the expression levels of MMP-2 and -9 in gelatin zymography, RT- PCR and western blot analysis without any cytotoxic influence. CM-chitin exhibited a higher MMP inhibitory effect than CM-chitosan through transcriptional down-regulations of activator protein-1 (AP- 1) and nuclear factor kB (NF-kB). Findings from the present study underscore the nutraceutical value of CM-chitosan and CM-chitin as a potent antioxidant and MMP inhibitor via alleviations of radical- induced oxidative damage. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding author at: Marine Bioprocess Research Center and Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea. Tel.: +82 51 629 7094; fax: +82 51 629 7099. E-mail address: sknkim@pknu.ac.kr (S.-K. Kim). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.09.004