Differential effects of allyl sulfides from garlic essential oil on cell cycle regulation in human liver tumor cells

Abstract

In this study, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), which are major organosulfur compounds (OSCs) of garlic, were used as experimental materials to investigate their modulation effects on cell viability and cell cycle in human liver tumor cells (J5). According to the results of cell viability assay, 50 or 100 μM DATS significantly decreased the cell viability as compared with the control (P < 0.05) in dose and time dependent relations. Phenomena of cell number loss, shape deformation and lysis were observed after treatment with 100 μM DATS for 24 h. Cell cycle studies showed that J5 cells were significantly arrested in G2/M phase as the cells were treated with 100 μM DADS, 10, 50 or 100 μM DATS for 24 h (P < 0.05). DATS was more effective in arresting cells in G2/M phase than DADS, and the phenomena of arresting J5 cells in G2/M phase increased obviously in dose and time dependent relations. According to the Western blot analysis, DATS decreased cyclin-dependent kinase (Cdks)-Cdk7 (i.e. Cdc2 activate kinase) protein levels in J5 cells but increased cyclin B1 protein level. The modulation potency to cyclin B1 and Cdk7 expressions was in the order of DATS > DADS > DAS. The modulation potency to cyclin B1 and Cdk7 protein levels increased with increasing in DATS concentration and culture time. In conclusion, DATS might affect cell viability and cell morphological changes in J5 cells and lead cells to be arrested in G2/M phase via controlling the expression of cyclin B1 and Cdk7 in J5 cells, and the controlling action might relate to the sulfuric atom numbers in the structures of all these allyl sulfides.

Introduction

Garlic (Allium sativum L.) is a widely consumed herb in foodstuffs and medicines. Epidemiological, clinical and laboratory studies have shown that crushed or processed garlic and their active principles, such as allicin, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), give diverse biological activities, including antitumorigenesis, antiatherosclerosis, blood sugar modulation and antibiotics (Milner, 2001; Siegel et al., 2004; Tsao and Yin, 2001; Kwon et al., 2003). Garlic has been reported to reduce chemically induced esophageal, skin, pulmonary, stomach, colon, mammary and lung tumor (Hong et al., 2000; Nakagawa et al., 2001; Takezaki et al., 2001; Iimuro et al., 2002; Ku et al., 2002). The anticarcinogenic/antitumorgenic effects of garlic have been attributed not only to the modulation of the antioxidation and/or drug-metabolizing enzyme system, but also to the reduction of cell proliferation and induction of apoptosis for tumor cells (Ledezma et al., 2004; Knowles and Milner, 2001; Oommen et al., 2004; Robert et al., 2001; Wu et al., 2001, Wu et al., 2002).

Human hepatocellular carcinoma (HCC) is one of the most common malignant tumors among human populations and a significant cause of mortality, especially in Taiwan. Thus, we are interested in the chemoprevention of garlic on liver cancer. Cancer is a complex and frightening disease. Many changes occur in cells as they become tumorigenic, including release from growth factor regulation, loss of contact inhibition, increased proteolysis, avoidance of immune surveillance, acquisition of immortality, promotion of angiogenesis and metastasis (Hartwell, 1997). It is classically defined as uncontrolled cellular division. The cell cycle is controlled by the temporally and spatially fluctuating activities of protein complexes (Murray and Hunt, 1993). Cell cycle progression requires the regulation of different cyclin, cyclin-dependent kinase (Cdk), Cdk inhibitor and dephosphorylation of several regulatory proteins (Morgan, 1995). Dietary components and medicines have been reported to significantly modify these regulators to blocking cells within the G0/G1, S, and G2/M phase of the cell cycle (Nakajima et al., 1996; Jeitner et al., 1998; Liang et al., 1999). Previous studies have shown the regulation of garlic and its active principles on cell proliferation, cell cycle regulation and apoptosis. Garlic oil, steam distillation product of fresh garlic, has a significant effect on arresting the G0/G1 phase of cell cycle of S-180 tumor cells (Xie et al., 1992). Allicin, the major source of OSCs of garlic essential oil, can inhibit the cell proliferation of erythroleukemia cell lines, HL-60 and K562, and arrest their cell cycle at S phase (Zheng et al., 1997). Allicin can also exhibit the antiproliferation effect on human mammary (MCF-7), endometrial (Ishikawa) and colon (HT-29) cancer cells, and accumulate cells at the G0/G1 and G2/M phases of the cell cycle in MCF-7 cells (Hirsch et al., 2000). In addition, DAS, DADS and DATS are produced from breakdown and rearrangement of allicin and they are the major components of garlic essential oil (Yu et al., 1989). Iciek et al. (2001) indicated that DADS could significantly inhibit the proliferation of HepG2 cells. DAS, DADS and DATS (100 μmol/l) significantly suppressed colon tumor cell proliferation (Knowles and Milner, 1998). DADS significantly increased the cell cycle arrest at G2/M phase of human colon tumor cells (HCT-15) via the inhibition of Cdc2 (cyclin-dependent kinase 1, Cdk1) activity (Knowles and Milner, 1998). The study about the effects of these allyl sulfides of garlic essential oil on cell cycle arrest and control factors of cell cycle is limited.

The garlic-rich OSCs are believed to play key roles in many biological effects. Among these OSCs, DAS, DADS and DATS, which differ in their number of sulfur atoms, are the three major constituents of garlic essential oil as shown in Fig. 1. The effect of OSCs from garlic essential oil on the antioxidation and drug-metabolizing system has attracted a great deal of investigation in our laboratory (Wu et al., 2001, Wu et al., 2002). Furthermore, we would like to investigate these three OSCs on cell viability and cell cycle of liver tumor cells (J5). In the present study, J5 cells were treated with various concentrations of DAS, DADS or DATS for various time periods, and the cell viability, cell cycle and the protein expression of cyclin and Cdk were investigated.