Chemico-Biological Interactions 182 (2009) 165–172 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Inhibition of proliferation of a hepatoma cell line by fucoxanthin in relation to cell cycle arrest and enhanced gap junctional intercellular communication Cheng-Ling Liu a , Yung-Sheng Huang a,1 , Masashi Hosokawa b , Kazuo Miyashita b , Miao-Lin Hu a,∗ a Department of Food Science, National Chung-Hsing University, 250 Kuo-Kuang Road, Taichung, 402, Taiwan b Faculty of Fisheries Science, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan article info Article history: Received 15 May 2009 Received in revised form 12 August 2009 Accepted 31 August 2009 Available online 6 September 2009 Keywords: Fucoxanthin Cytotoxicity Apoptosis Connexin 43 Connexin 32 Gap junctional intercellular communication abstract Fucoxanthin is one of the most abundant carotenoids found in Undaria pinnatifida and has been shown to inhibit tumor proliferation in vitro. However, the mechanisms underlying the anti-cancer effects of fucoxanthin are unclear. In this study, we hypothesized that fucoxanthin may cause cell cycle arrest and enhance gap junctional intercellular communication (GJIC) in SK-Hep-1 human hepatoma cells. Data revealed that fucoxanthin (1–20 M) strongly and concentration-dependently inhibited the proliferation of SK-Hep-1 cells at 24 h of incubation, whereas fucoxanthin facilitated the growth of a murine embry- onic hepatic (BNL CL.2) cells at 24 h of incubation and only slightly slowed the cell proliferation at 48 h. In SK-Hep-1 cells, fucoxanthin caused cell cycle arrest at G0/G1 phase and induced cell apoptosis, as evi- denced by increased subG1 cells and induction of DNA strand breaks. Using scrape loading-dye-transfer assay, fucoxanthin was found to significantly enhance GJIC of SK-Hep-1 cells without affecting that of BNL CL.2 cells. In addition, fucoxanthin significantly increased protein and mRNA expressions of connexin 43 (Cx43) and connexin 32 (Cx32) in SK-Hep-1 cells. Moreover, fucoxanthin markedly increased the concen- tration of intracellular calcium levels in SK-Hep-1 cells. Thus, fucoxanthin is specifically antiproliferative against SK-Hep-1 cells, and the effect is associated with upregulation of Cx32 and Cx43, which enhances GJIC of SK-Hep-1 cells. The enhanced GJIC may be responsible for the increase of the intracellular calcium level, which then causes cell cycle arrest and apoptosis. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Epidemiological studies suggest a correlation of elevated intakes of fruits and vegetables rich in carotenoids with decreased risks of cancer [1]. The total production of carotenoids in nature has been estimated at 100 million tones per year [2]. Among them, fucoxan- thin, having a unique structure including an unusual allenic bond and 5,6-monoepoxide in its molecule, is the most abundant of all carotenoids accounting for >10% of estimated total natural pro- duction of carotenoids [3]. Studies have shown that fucoxanthin induces apoptosis in prostate cancer cells [4], leukemia cells [5] and colon cancer cells [6]. In addition, fucoxanthin has been shown to cause cell cycle arrest in neuroblastoma cells [7] and human hepatoma HepG2 cells [8]. Gap junctions are intercellular membrane channels between adjacent cells that are formed by two hemichannels each con- Abbreviations: GJIC, gap junctional intercellular communication; Cx32, connexin 32; Cx43, connexin 43. ∗ Corresponding author. Tel.: +886 4 2281 2363; fax: +886 4 2281 2363. E-mail address: mlhuhu@dragon.nchu.edu.tw (M.-L. Hu). 1 Y.-S Huang contributed equally as the corresponding author. sisting of six connexin protein. Much evidence indicates that gap junctional intercellular communication (GJIC) plays an important role in maintaining homeostasis in multicellular organs. The role of GJIC in carcinogenesis is primarily based on actions such as inhibi- tion of tumor promoters [9] and aberrant communication in tumor cells [10]. Presently, at least 21 members of connexin gene have been identified; in particular, connexin 32 (Cx32) and connexin 43 (Cx43) are the major kinds of gap junction proteins express- ing in hepatocytes [11,12]. Mutations or deficiencies in Cx genes have recently been related to several diseases including cancer [13]. Connexin proteins may be degraded by lysosomes and proteo- somes in most tumors [14]. Studies have shown that expressions of Cx32 and Cx43 proteins are decreased in hepatocellular carci- noma tissues [15–17]. Importantly, connexins may be considered as tumor-suppressors [18]. For instance, it has been shown that transfection of Cx genes to human glioblastoma cells significantly reduces cell proliferation [19]. In addition, Cx43 has been shown to reduce levels of S phase kinase-associated protein 2, which sup- presses degradation of p27, a key protein in cell cycle between the G1 and S phases [20]. Because the mechanisms by which fucoxanthin causes apop- tosis and cell cycle arrest in cancer cells are unclear, the present study aimed to investigate whether the inhibitory effect of fucoxan- 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.08.017