Introduction

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Numerous epidemiological studies revealed that high consumption of fruits, vegetables, and other plant products may reduce the incidence and development of colorectal cancer (Tuyns et al., 1988; Steinmetz and Potter, 1991; Steinmetz et al., 1994). Since colorectal cancers are difficult to treat with existing therapeutic modalities, identifying dietary phytochemicals that have antitumor activity and investigating their mechanisms of action may lead to significant advances in the prevention of human cancer (Block et al., 1992). The monoterpenes, found in essential oils of citrus fruits, cherry, mint, and herbs, are non-nutritive dietary microconstituents mainly responsible for the distinctive fragrance of many plants. They are used as flavor additives in food, beverages, and perfumes.

Recents studies have shown that monoterpenes exert antitumor activities and suggest that these components are a new class of cancer chemopreventive agents (Elson and Yu, 1994; Kelloff et al., 1996; Crowell, 1999). Limonene, a main constituent of orange and citrus peel oils, has been reported to exert antitumor activity against mammary gland, lung, liver, stomach, and skin cancers in rodents (Elegbede et al., 1986; Wattenberg and Coccia, 1991; Crowell and Gould, 1994; Mills et al., 1995; Kawamori et al., 1996). Similarly, perillyl alcohol, a hydroxylated limonene analog, exhibits chemopreventive activity against liver, mammary gland, pancreas, and colon cancers in rodents (Haag and Gould, 1994; Stark et al., 1995; Reddy et al., 1997). More recently, geraniol, an acyclic monoterpene alcohol found in lemongrass and aromatic herb oils, has been shown to exert in vitro and in vivo antitumor activity against murine leukemia, hepatoma, and melanoma cells (Shoff et al., 1991; Yu et al., 1995; Burke et al., 1997).

No information is available on the potential effects of geraniol on colon cancer. Therefore, we examined its effect on Caco-2 cell growth, a human colonic cancer cell line. We also measured the effect of geraniol on polyamine metabolism, which is known to be enhanced in cancer cells and which might be one of the targets of the chemopreventive action of geraniol (Seiler et al., 1998).

	Materials and Methods

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Cell Culture. Caco-2 cells were obtained from the European Collection of Animal Cell Culture (CERDIC, Sophia Antipolis, France) and were cultured in 75-cm² Falcon flasks containing Dulbecco's modified Eagle's medium (DMEM) at 25 mM glucose supplemented with 10% heat-inactivated horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ and subcultured after trypsinization (0.5% trypsin/2.6 mM EDTA). They were used up to 30 to 40 passages.

Cell Growth. Cells were seeded in 96-well plates and incubated for different times. Cell growth was stopped by addition of 50 µl of trichloroacetic acid (50% v/v), and protein content of each well was determined by staining with sulforhodamine B (Skehan et al., 1990). Absorbance was determined at 490 nm. The relationship between cell number (protein content per well) and absorbance is linear from 0 to 200,000 cells.

Cell Cycle Analysis. Cells (3.5 × 10⁵ per 10-ml Petri dish) were treated with geraniol (400 µM) or 0.1% ethanol 24 h after seeding in supplemented DMEM culture medium. Control and geraniol-treated cells were harvested by trypsinization (0.5% trypsin/2.6 mM EDTA) from day 4 to day 6, and washed twice with ice-cold PBS and fixed in methanol/PBS (9:1, v/v) at 20°C for at least 30 min. The fixed cells were then washed twice with ice-cold PBS and stained with 50 µg/ml of propidium iodide in the presence of 25 µg/ml of RNase A. Cell cycle phase distribution was analyzed in three different experiments using flow cytometry (FACS scan flow cytometer, Becton Dickinson Immunocytometry Systems, San Jose, CA) (Nicoletti et al., 1991). Data from 10,000 events per sample were collected and analyzed using the Cell Fit cell analysis program (Becton Dickinson).

Measurement of de novo DNA Synthesis. Cells were treated with geraniol (400 µM) or 0.1% ethanol for 4 days and were then exposed to 0.8 µCi/ml methyl-[³H]thymidine (3 TBq/mmol; Amersham Pharmacia Biotech, Orsay, France) for 4 h. Cells were harvested by scraping and then sonicated. The radioactivity present in the trichloracetic-acid-precipitable material was determined by liquid scintillation spectrometry.

Determination of Cell Apoptosis and Cytotoxicity. Cells (7.10⁵ per 10-ml Petri dish) were seeded and treated with 400 µM geraniol for 24, 30, and 48 h. After trypsinization, cells were collected by centrifugation and stored at 80°C. Apoptotic DNA was separated from genomic DNA, using the Suicide Track DNA ladder kit (Oncogene Research Products, Cambridge, MA). The DNA fragments were separated by electrophoresis and stained with ethidium bromide.

Determination of Intracellular Polyamines. Cells (6.10⁵) were seeded in 10-ml Petri dishes and incubated in supplemented DMEM culture medium. Twenty-four hours after seeding, cells were incubated with geraniol (400 µM) or 0.1% ethanol for 1 day. Cell layers were washed with versene (40 mM NaCl, 3 mM KCl, 1.5 mM KH₂PO₄, 15 mM Na₂HPO₄, 2.6 mM EDTA; pH 7.2), harvested by scraping and pelleted by centrifugation. The cell pellets were homogenized in perchloric acid (200 mM), and the homogenates were centrifuged at 3000g for 10 min after standing for 16 h at 2°C. The acid-insoluble pellets were used for protein determination, and the clear supernatants were applied on a reversed phase column for separation. The polyamines (putrescine, spermidine, and spermine) were determined by separation of the ion pairs formed with n-octanesulfonic acid, reaction of the column effluent with o-phthalaldehyde/2-mercaptoethanol reagent, and monitoring of fluorescence intensity (Seiler and Knödgen, 1980).

Ornithine Decarboxylase (ODC) and S-Adenosylmethionine Decarboxylase (AdoMetDC) Activities. Cells were homogenized in 100 mM Tris-HCl buffer, pH 7.4 (1 mM EDTA, 1 mM dithiothreitol, 0.5 µM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride). After centrifugation at 33,000g for 25 min at 4°C, ODC and AdoMetDC assays were performed in the supernatants. ODC activity was measured by the rate of ¹⁴CO₂ formation from [1-¹⁴C]L-ornithine (55 mCi/mmol, Amersham Pharmacia Biotech) (Richman et al., 1971) and AdoMetDC activity by measuring the rate of ¹⁴CO₂ formed from [1-¹⁴C]S-adenosylmethionine (60 mCi/mmol, Amersham Pharmacia Biotech) (Richman et al., 1971).

Statistical Analysis. Data are reported as means ± S.E. Statistical differences between control and geraniol-treated cells were evaluated using the Student's t test. Differences were considered significant at p < 0.05.

	Results

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Geraniol and Caco-2 Cell Growth. The dose-response effect of geraniol on cell proliferation was studied at concentrations between 100 and 500 µM. As shown in Fig. 1, geraniol inhibited the proliferation of Caco-2 cells in a dose-dependent manner. At 400 µM, geraniol caused a 70% inhibition of cell growth. This concentration was used in all further experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Caco-2 cell growth curves. Cells were seeded at 4500 cells in 96-well plates in DMEM supplemented with 3% horse serum, transferrin (5 µg/ml), selenium (5 ng/ml), and insulin (10 µg/ml). Geraniol was added at different concentrations 24 h after seeding. The culture medium (containing 0.1% ethanol, with and without geraniol) was replaced every 24 h. Values represent means ± S.E. (n = 8).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Flow cytometry analysis of Caco-2 cells. Cells were treated with or without 400 µM geraniol for 6 days. Cells were harvested from day 4 to day 6 and analyzed by flow cytometry in three separate experiments. The data show the distribution of the cells in the G1, G2-M, and S phases of the cell cycle. Values represent means ± S.E. (n = 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Incorporation of methyl-[3H]thymidine into DNA. Cells were maintained for 4 days in culture in the absence (open column) or in the presence (hatched column) of 400 µM geraniol, and then treated with 0.8 µCi/ml methyl-[3H]thymidine for 4 h. The radioactivity present in the trichloracetic-acid-precipitable materials was determined by liquid scintillation spectrometry. Values represent means ± S.E. (n = 3); **p < 0.01.

Effects of Geraniol on Apoptosis and Cytotoxicity. A potential cytotoxic mechanism of geraniol is the induction of apoptosis. This possibility was tested by DNA fragmentation assays. Cells treated with geraniol did not exhibit apoptotic DNA fragmentation ladders (results not shown). This result is confirmed by the absence of a sub-G₁ peak as shown by flow cytometry analysis.

Effects of Geraniol on Polyamine Metabolism. In Caco-2 cells treated with geraniol (400 µM), a 50% diminution of ODC activity was observed after 6 and 16 h of treatment (Fig. 4). In contrast, geraniol treatment led to a significant increase in AdoMetDC activity at 16 and 24 h by 20 to 30%, respectively.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   ODC and AdoMetDC activities (picomoles CO2 per milligrams of protein per hour) in Caco-2 cells maintained for 6, 16, and 24 h in culture in the absence (open columns) or in the presence (hatched columns) of 400 µM geraniol. The culture medium (containing 0.1% ethanol, with and without geraniol) was replaced every 24 h. Results are means ± S.E. of three separate experiments; *p < 0.05.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Effect of geraniol (400 µM) on the polyamine content (picomoles per milligrams of protein) of Caco-2 cells cultured for 16, 24, and 48 h in the absence (open columns) or in the presence (hatched columns) of 400 µM geraniol. The culture medium (containing 0.1% ethanol, with and without geraniol) was replaced every 24 h. Results are means ± S.E. of three separate experiments; *p < 0.05.

	Discussion

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In the present study we demonstrate that the dietary monoterpene geraniol inhibits the growth of human colon cancer cells. It was previously reported that geraniol inhibited the growth of leukemia and melanoma cells (Shoff et al., 1991), hepatoma cells (Yu et al., 1995), and pancreatic cancer cells (Burke et al., 1997). We show that the antiproliferative effects of geraniol on human colon cancer cells are related to its ability to decrease the rate of DNA synthesis, which affects progression of the cells through the S phase of the cell cycle. Flow cytometric analyses and the absence of DNA fragmentation showed that geraniol does not induce apoptosis of Caco-2 cells. Furthermore, the compound has no cytotoxic properties, indicating that geraniol exerts mainly cytostatic effects on human colon cancer cells.

The effects of geraniol on the cell cycle differ from those described for another monoterpene, perillyl alcohol, which caused a considerable decrease in the percentage of human breast cancer cells engaged in the S phase, with a corresponding increase of the proportion of cells in G₁ and a decrease in cyclin D1 expression (Bardon et al., 1998). The antiproliferative effects of geraniol on hepatoma and melanoma cell growth have been ascribed to inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, a key enzyme of mevalonate biosynthesis (Elson, 1995). Inhibition of HMG-CoA reductase by geraniol causes a reduction of the mevalonate pool, and thus limited protein isoprenylation, which involves the post-translational covalent attachment of a lipophilic farnesyl or geranylgeranyl isoprenoid group to numerous proteins (Clarke, 1992). Many prenylated proteins, like Ras, the lamins A and B, and Rho regulate cell growth and/or transformation (Crowell, 1999). It was reported that inhibition of HMG-CoA reductase in C6 glioma cells provokes inhibition of DNA synthesis and that prenylation of proteins is essential for progression of cells into the S phase (Crick et al., 1998). In the present report, we show that geraniol caused inhibition of DNA synthesis, which was accompanied by the accumulation of Caco-2 cells in the S phase.

Our study shows that polyamine metabolism could be another target of the antitumor properties of geraniol. A high expression of ODC is characteristic for tumor cells (Pegg, 1988). Geraniol caused a rapid decrease of ODC activity in Caco-2 cells and a significant decrease of the intracellular putrescine content. In addition, geraniol caused an important accumulation of N¹-acetylspermidine, indicating a high polyamine acetylation rate. This is related to increased polyamine catabolism and elimination. It seems to correlate with the reduced amount of spermine in geraniol-treated cells, despite the increased activity of AdoMetDC, the enzyme involved in spermidine and spermine synthesis. The higher level of AdoMetDC activity in the geraniol-treated cells may be due to the feedback control of this enzyme by spermine (Duranton et al., 1998).

ODC expression varies considerably during progression of cells through the cell cycle, with peaks in the early S phase (Cohen, 1998). ODC inhibition by geraniol in Caco-2 cells may explain the accumulation of cells in the S phase. The reduction of ODC activity by geraniol might be caused by the impairment of the prenylation of Ras in geraniol-treated Caco-2 cells, since it was shown that cells overexpressing Ras have constitutively high levels of ODC activity that correlate with oncogenic transformation (Shantz and Pegg, 1998). In addition, Raf activation of ODC expression is mediated by tandem E-boxes contained in the first intron of the ODC gene (Aziz et al., 1999).

In conclusion, our results are the first to describe a potent antiproliferative effect of geraniol on the growth of human colon cancer cells. Geraniol has no cytotoxic effect, is mainly cytostatic, and inhibits DNA synthesis, leading to the accumulation of Caco-2 cells in the S phase. Inhibition of ODC expression may be one of several targets involved in the antiproliferative effects of geraniol. However, it remains to be determined whether polyamine depletion by itself is directly responsible for the observed antiproliferative effect. The low toxicity of geraniol makes it attractive for in vivo studies in colon cancer prevention and treatment.