Growth inhibition of human hepatic carcinoma HepG2 cells by fucoxanthin is associated with down-regulation of cyclin D

https://doi.org/10.1016/j.bbagen.2008.01.003Get rights and content

Abstract

Fucoxanthin, a major carotenoid in brown sea algae, has recently been demonstrated by us to inhibit the proliferation of colon cancer cells, and this effect was associated with growth arrest. These results, taken together with previous studies with fucoxanthin, suggest that it may be useful in chemoprevention of other human malignancies. The present study was designed to evaluate the molecular mechanisms of fucoxanthin against hepatic cancer using the human hepatocarcinoma HepG2 cell line (HepG2). Fucoxanthin reduced the viability of HepG2 cells accompanied with the induction of cell cycle arrest during the G0/G1 phase at 25 μM. This concentration of fucoxanthin inhibited the phosphorylation of the retinoblastoma protein (Rb) at Serine 780 (Ser780) position 18 h after treatment. The kinase activity of cyclin D and cdk4 complex, responsible for the phosphorylation of Rb Ser780 site, was down-regulated 18 h after the treatment. Western blotting analysis revealed that the expression of cyclin D-type protein was suppressed by treatment of fucoxanthin. This reduction was partially blocked by concurrent treatment with the proteasome inhibitor MG132, indicating the involvement of the proteasome-mediated degradation. In addition, RT-PCR analysis revealed that fucoxanthin also appeared to repress cyclin D mRNA. Thus, both the protein degradation and transcriptional repression seem to be responsible for suppressed cyclin D level in fucoxanthin-treated HepG2 cells which may be related to the antitumorgenic activity.

Introduction

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer mortality and the third most common cause of cancer-related death [1]. Most of the cases of hepatic cancer are not curable due to extensive liver dysfunction caused by concomitant cirrhosis, infrequent diagnosis at an early stage, and lack of appropriate chemotherapy. However, a number of epidemiological studies demonstrated that the risk of liver cancer might be reduced by the consumption of vegetables [2], [3], [4]. Among the various components of vegetables, carotenoids are one of the major active compounds, which can prevent different types of human malignancies like colon [5], [6], lung [7], and breast [8] cancer. Regarding HCC, protective role of carotenoids have also been reported. In a cohort study, lower plasma levels of β-carotene, the most widely studied carotenoid, appeared to be more predictive of elevated HCC risk associated with smoking and alcohol-drinking groups [9]. In addition, treatment with β-carotene has been shown to reduce the incidence of diethylnitrosamine-initiated and phenobarbital-promoted hepatocarcinogenesis in an animal model [10]. Other carotenoids like canthaxanthin, astaxanthin, lutein, and lycopene are also capable of suppressing carcinogen-induced HCC in animal models [11], [12], [13].

The growth of cells is normally determined by extracellular signals that control the gene expression and protein regulation required for cell division [14]. In contrast, during tumor progression, cancer cells are conferred with the capacity to proliferate independently of exogenous growth-promoting or growth-inhibitory signals [15], [16]. Thus, the antiproliferative effect of chemicals or drugs on cancer cells is one of the mechanistic ways to exert their anticarcinogenic activity. A number of compounds have shown their antineoplastic effects on HCC by inducing cell cycle arrest or apoptosis. For example, acyclic retinoid, a derivative of retinoid used for several clinical trials [17], [18], inhibits proliferation by inducing cell cycle arrest in hepatic carcinoma cells [19]. Cisplatin and methoxymorpholinyl doxorubicin, clinically used as chemotherapeutic drugs against several cancers, proved preventive effect against HCC by inhibiting cellular proliferation [20], [21].

Fucoxanthin, whose structure is shown in Fig. 1, is an oxygenated carotenoid available in different types of edible seaweed such as Laminaria japonica, Undaria pinnatifida, and Hijikia fusiformis. Many biological functions of this compound have been studied; e.g., suppressive effect on adipocyte differentiation [22], antimutagenicity [23], anti-ocular inflammation [24], and cancer preventing effects. In our previous study [25], fucoxanthin exerted an antiproliferative effect by inducing cell cycle arrest at G0/G1 phase in human colon carcinoma cells. The oral administration of fucoxanthin suppressed the development of aberrant crypt foci, a pre-neoplastic marker for colon neoplasia, in azoxymethane-treated mice [26], and also showed chemopreventive effects against N-ethyl-N′-nitro-N-nitrosoguanidine-induced mouse duodenal carcinogenesis [27], two-stage mouse skin carcinogenesis [23], and 1,2-dimethylhydrazine-induced colon carcinogenesis [28]. Concomitantly, this compound has been shown to have growth-inhibitory effects on various cell lines; e.g., prostate cancer PC-3, DU 145, and LNCaP cells [29], leukemia HL-60 cells [30], colon cancer HT-29, Caco-2, and DLD-1 cells [31], and neuroblastoma GOTO cells [32]. As hepatocarcinogenesis is concerned, fucoxanthin suppressed the spontaneous tumorigenesis in the liver of C3H/He male mice [23]. However, the mechanism by which fucoxanthin exerts these anticarcinogenic effects is unknown.

In the present study, hepatocarcinoma HepG2 cells were employed to elucidate the preventative mechanism of fucoxanthin against HCC.

Section snippets

Materials

Propidium iodide (PI) and RNase A were purchased from Sigma Chemical (St. Louis, MO). For cell culture, Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo, Japan), and fetal bovine serum (FBS) was from Sigma Chemical. A proteasome inhibitor, MG132, and a fluorogenic peptide substrate, succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), were purchased from Peptide Institute, Osaka, Japan. All other reagents

Fucoxanthin inhibits the growth of human hepatoma cells through cell cycle arrest at G0/G1 phase

The effects of fucoxanthin on cell viability in HepG2 cells were investigated by an MTS assay (Fig. 2A). Fucoxanthin dose-dependently suppressed cell growth at concentrations of 10, 25 and 50 μM 72 h after treatment, the end time point of this study compared with vehicle alone (0.5% THF). However, at 24 h, only 25 and 50 μM fucoxanthin showed a significant growth inhibition. In a cell population, cell growth is the process deriving from the balance of cell proliferation and cell death. Then,

Discussion

The present results characterized the growth-inhibitory effect of fucoxanthin on the human hepatic cancer cell line. The effect was primarily due to an arrest in the G0/G1 phase of the cell cycle and apoptosis was not observed under the present condition. It is likely that the fucoxanthin possesses cytostatic rather than cytocidal activity in this cells and then we further investigated the mechanisms of cell cycle regulation. The phosphorylation of Rb plays a crucial role in the progression of G

Acknowledgments

This research was supported by the Research and Development Program for New Bio-industry Initiatives (2006–2010) of Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.

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