Inhibitory effect of gingerol on the proliferation and invasion of hepatoma cells in culture
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- Yagihashi, S., Miura, Y. & Yagasaki, K. Cytotechnology (2008) 57: 129. doi:10.1007/s10616-008-9121-8
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Effect of -gingerol, a major pungent component in ginger, on the proliferation of a rat ascites hepatoma AH109A cells was investigated by measuring [3H]thymidine incorporation into acid-insoluble fraction of the cultured cells and that on the invasion by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells. -Gingerol inhibited both the proliferation and invasion of hepatoma cells in a dose-dependent manner at concentrations of 6.25–200 μM (proliferation) and 50–200 μM (invasion). -Gingerol accumulated cells in S phase and elongated doubling time of hepatoma cells, and increased the rate of apoptosis. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. -Gingerol suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with -gingerol, HX and XO or with -gingerol and hydrogen peroxide. Furthermore, -gingerol reduced the intracellular peroxide levels in AH109A cells. These results suggest that the suppression of hepatoma cell proliferation by -gingerol may be due to cell cycle arrest and apoptosis induction. They also suggest that the anti-oxidative property of -gingerol may be involved in its anti-invasive activity of hepatoma cells.
KeywordsAnti-oxidative propertyApoptosisCell cycle-GingerolHepatoma cellsHydrogen peroxideInvasionProliferationReactive oxygen species
Eagle’s minimum essential medium
Reactive oxygen species
-Gingerol, a major pungent component in ginger has been shown to possess various pharmacological activities such as anti-oxidative and anti-inflammatory activities (Surh 2002; Wang et al. 2003). The latter is due to the inhibitory effect of -gingerol on the expression of cyclooxygenase 2, a key enzyme in the prostaglandin biosynthesis and recognized as a molecular target for anti-inflammatory and cancer chemopreventive agents (Kim et al. 2004). -Gingerol has been reported to induce apoptosis in HL60 cells (Lee and Surh 1998) and inhibit metastasis of B16 melanoma cells (Suzuki et al. 1997). The invasion is particularly complicated process and key step in the cancer metastatic cascade (Liotta et al. 1988). During the past 20 years, primary liver cancer, 95% of which is hepatocellular carcinoma, has ranked third in men and fifth in women as a cause of death from malignant neoplasm in Japan (Kiyosawa et al. 2004). AH109A cells, a rat ascites hepatoma line, grow rapidly both in vitro and in vivo, and they form solid tumors when subcutaneously inoculated into rats. Using AH109A cells, we have demonstrated that some food factors such as carotenoids (Kozuki et al. 2000), catechins (Zhang et al. 2000), resveratrol (Kozuki et al. 2001), ascorbic acid (Hirakawa et al. 2005), lignans (Miura D et al. 2007) suppress the proliferation and/or invasion of the hepatoma cells. Furthermore, we have confirmed an involvement of reactive oxygen species (ROS) in hepatoma cell invasion: both the exogenous and endogenous ROS induce the invasion of AH109A cells (Kozuki et al. 2000; Miura Y et al. 2003a). The present study was attempted to investigate the effect of -gingerol on the proliferation and invasion of AH109A cells in culture and its modes of actions.
Materials and methods
-Gingerol was purchased from Nakalai Tesque, Inc., Kyoto, Japan. It was dissolved in ethanol and the solution was added to the medium at a final ethanol concentration of 0.2%. Control medium contained 0.2% ethanol alone. All other reagents were of the best grade commercially available.
Culture of AH109A hepatoma cells
The experiments in this article were conducted in accordance with guidelines established by the Animal Care and Use Committee of Tokyo Noko University. Male Donryu rats (4 weeks of age) were purchased from NRC Haruna (Gunma, Japan). AH109A cells were generously provided by the Cell Resource Center for Biomedical Research, Tohoku University, Sendai, Japan. AH109A cells were maintained in peritoneal cavity of male Donryu rats, and isolated from accumulated ascites and then cultured in Eagle’s minimum essential medium (MEM) (Nissui Pharmaceutical Co., Tokyo, Japan) containing 10% calf serum (CS, Equitech-Bio, Inc., Kerrville, TX, USA) (10% CS/MEM). These cells were cultured for at least 2 weeks after isolation to eliminate contaminated macrophages and neutrophils, and used for the assays described below.
In vitro proliferation and invasion assays
Effect of -gingerol on the proliferation was examined by measuring the incorporation of [methyl-3H]thymidine (20 Ci/mmol, Perkin Elmer Life and Analytical Sciences, Inc., Boston, MA, USA) into acid-insoluble fraction of cells, as described previously (Yagasaki et al. 1992). Briefly, 1.0 × 104 AH109A cells were seeded into a 48-well plate and exposed to -gingerol for 24 h in 400 μL of 10% CS/MEM. The proliferation of AH109A cells was evaluated by measuring the incorporation for last 4 h of [methyl-3H]thymidine (0.15 μCi/well) into the DNA fraction. Effect of -gingerol on the invasion was examined by the co-culture system (Akedo et al. 1986) with slight modifications as described previously (Kozuki et al. 2000). Briefly, mesothelial cells (M-cells) were isolated from mesentery of male Donryu rats (6–8 weeks of age). After digestion by trypsin, 1.3–2.0 × 105 M-cells were plated in a 60 mm culture dish with 2 mm grids (Corning Incorporated, Corning, NY, USA), and cultured for 5–7 days to attain a confluent state in 10% CS/MEM. Then, AH109A cells (2.4 × 105 cells per dish) were applied on the monolayer of M-cells in 10% CS/MEM with -gingerol for 24 h. Invaded cells and colonies underneath M-cells were counted with a phase-contrast microscope. The invasive activity of AH109A cells was expressed as the number of invaded cells and colonies/cm2.
Flowcytometric analyses of cell cycle phases and Annexin-V-FITC staining of apoptotic cells
For cell cycle analysis, 2.5 × 105 cells of AH109A per well were seeded in a 6-well plate in the medium containing 0, 25, 50, 100, and 200 μM -gingerol and cultured for 24 h. Cells were collected and washed twice with sterilized, Ca2+- and M2+-free, phosphate-buffered saline [PBS(−)]. Thereafter, 500 μL of propidium iodide (PI) solution containing 1 mg of PI (Sigma Chemical Co., St. Louis, MO, USA) in 20 mL of 1% Triton X-100 (Sigma), and 0.1% of sodium citrate (Wako Pure Chemical Industries Ltd., Osaka, Japan) was added and cells were incubated for 30 min on ice. Cells at different cell cycle phases were then analyzed with a flow cytometer (EPICS ELITE EPS, Beckman-Coulter, Hialeah, FL, USA) as previously described (Zhang et al. 2000).
The effect of -gingerol on apoptosis in AH109A cells was assessed using ANNEXIN-V FITC kit (IMMUNOTECH, Marseille, France) according to the manufacturer’s instructions. Phosphatidylserine (PS), which can specifically bind to Annexin-V, is one of the phospholipids in the cell membrane and exists predominantly in inner leaflet of the cell membrane of normal cells. When apoptosis occurs, PS in the cell membrane immediately appears on the outer leaflet of the cell membrane. The cells with PS on their surface can thus be thought to be early apoptotic cells. Briefly, 5 × 105 cells of AH109A per well were seeded in a 6-well plate and cultured in the medium containing 0, 100, and 200 μM -gingerol for 3 h. At the end of culture, cells were labeled with Annexin-V-FITC and analyzed with a flow cytometer as described previously (Miura Y et al. 2004).
Pretreatment of AH109A cells with hypoxanthine and xanthine oxidase or hydrogen peroxide
AH109A cells were cultured for 4 h in the absence or presence of 20 μM -gingerol with or without a ROS-generating system, i.e., 4 μg/mL hypoxanthine (HX, Sigma, St. Louis, MO) with 7 × 10−4 U/mL xanthine oxidase (XO, Sigma) (Shinkai et al. 1986; Tanaka et al. 1997) or 25 μM hydrogen peroxide (H2O2, Wako Pure Chemical Industries). AH109A cells were then washed once with 10% CS/MEM and seeded on the M-cell monolayer in 10% CS/MEM without -gingerol and ROS. After cultured for 24 h, invaded cells and colonies underneath M-cells were counted with a phase-contrast microscope as described above.
Flowcytometric analysis of intracellular peroxide in AH109A cells
Data were expressed as means ± SEM. Multiple comparison was performed by one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test, and p < 0.05 was considered statistically significant.
Effect of -gingerol on the proliferation and invasion of AH109A cells
Accumulation in S phase and induction of apoptosis in -gingerol-treated AH109A cells
Effect of -gingerol on the invasion of AH109A cells pre-treated with hypoxanthine and xanthine oxidase or hydrogen peroxide
Intracellular peroxide levels of AH109A cells pre-treated with hypoxanthine and xanthine oxidase or hydrogen peroxide
In this study, -gingerol was demonstrated to inhibit both the proliferation and invasion of AH109A cells in vitro (Fig. 1). -Gingerol concentrations that could significantly suppress the proliferation of AH109A cells (Fig. 1a, 12.50–200 μM) were lower than those which could suppress the invasion of AH109A cells (Fig. 1b, 50–200 μM). -Gingerol commenced to significantly inhibit the hepatoma proliferation at a concentration of 12.5 μM and almost completely inhibited (6% of control) at 200 μM (Fig. 1a), whereas it significantly inhibited the invasion at 50 μM, which was four times as high as 12.5 μM, and the highest inhibition observed at 200 μM was 34% of control (Fig. 1b). These results suggest that the inhibition of proliferation by -gingerol might not be a main cause of its inhibitory action against the invasion.
To elucidate the mechanisms of inhibition of hepatoma cell proliferation by -gingerol, the effect of -gingerol treatment on cell cycle and on the rate of apoptosis was examined by using flow cytometry. -Gingerol was found to suppress proliferation from lower concentrations (25 μM∼) by increasing doubling time of AH109A cells through accumulation in the S phase (Fig. 2). Further, -gingerol increased the rate of apoptosis at higher concentrations (100–200 μM) (Fig. 3). -Gingerol has been reported to induce apoptosis in HL60 cells (Lee and Surh 1998). It is apparent that different cell lines have different sensitivities to -gingerol but the primary target in cell cycle and apoptosis may be the same in these cell lines. Although the doses of -gingerol which induced apoptosis were higher than those which induced cell cycle arrest in AH109A hepatoma cells, these results suggest that -gingerol affects both cell proliferation and cell death which at least partly account for the inhibitory effect of -gingerol on the proliferation of AH109A cells.
Our previous works have demonstrated that the invasion of AH109A cells is accelerated by ROS (Kozuki et al. 2000). In the present study, we therefore examined the effect of -gingerol on the ROS-potentiated invasive activity using both HX–XO system and H2O2. -Gingerol was found to inhibit the ROS-induced elevation of the AH109A invasion (Fig. 4). -Gingerol was also found to scavenge intracellular peroxides (Fig. 5). We have found that ROS can induce gene expression of hepatocyte growth factor (HGF), which is known as a cell motility factor (Parr and Jiang 2001), in M-cells as well as AH109A cells (Miura Y et al. 2003b). Thus, HGF produced by both AH109A and M-cells may potentiate the motility of AH109A cells and also may induce the retraction of M-cells, leading to acceleration of the AH109A invasion. Provided that -gingerol, like a polyphenol resveratrol (Miura D et al. 2004), suppresses the production of HGF through its anti-oxidative activity, -gingerol may diminish the induction of the retraction of M-cells as well as the motility of AH109A cells, this leading to the effective reduction of the AH109A invasion by reducing the functions of both cells at the same time. Since prostaglandins are shown to enhance the invasion of hepatoma cells (Miura D et al. 2003), a possibility that -gingerol, a cyclooxygenase inhibitor (Kim et al. 2004), interrupts prostaglandin synthesis and hence inhibits the invasion cannot be ruled out. Lee et al. have recently reported that -gingerol inhibits cell adhesion, invasion, motility and activities of matrix metalloproteinase (MMP)-2 and 9 in MDA-MB-231 human breast cancer cells (Lee et al. 2007). AH109A cells are different from this breast cancer cells, for instance, in the productivity of MMPs; AH109A cells do not produce MMPs. Thus, different action sites as well as common action sites of -gingerol may be existent between these two cancer cells. Further studies are required to clarify these aspects.
In summary, we clearly demonstrated that -gingerol inhibited the proliferation and invasion of AH109A hepatoma cells in culture, the proliferation being more strongly suppressed than was the invasion. -Gingerol induced cell cycle arrest at lower concentrations and apoptosis at higher concentrations in the hepatoma cells. It also suppressed the ROS-induced increases in invasive capacity and intracellular peroxide levels. These results suggest that -gingerol affects both cell proliferation and cell death that account for, at least partly, the inhibitory effect of -gingerol on the proliferation of AH109A cells. They also suggest that the anti-oxidative property of -gingerol may be involved in its anti-invasive action. -Gingerol may have promising beneficial effects in preventing tumor growth and metastasis and may be of significance from the aspect of nutritional control of cancers.
This work was supported by the Fund from the Yamazaki Spice Foundation, Tokyo, Japan.