MKN-45 and MKN-28, human gastric cancer cell lines, were obtained from Korean Cell Line Bank (Seoul, Korea). All cells were grown in RPMI 1640 (Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum in a humidified 5% CO₂ at 37°C containing 50 mg/ml gentamicin, and 135 mg/ml glutamine. The medium was changed three times per week. The number of cells was counted with a hematocytometer. Urushiol complex was obtained in a solution form from Lifetree Biotech Co., Ltd. (Suwon, Korea). The aqueous solution was filtered through Whatman No. 2 filter paper, and then concentrated in vacuum to dryness. The yield of urushiol was about 3.5%. The powder was weighted and adjusted to a final concentration with DMSO (Sigma-Aldrich, St. Louis, MO, USA) and stored at ?20°C until use. The stock solution of urushiol was diluted with medium to the desired concentration prior to use. The maximal concentration of DMSO did not exceed 0.1% (v/v) in the treatment range (2?10 μg/ml), where there was no influence on the cell growth (data not shown).

The proliferation of gastric cancer cells was assessed by using the MTT assay which is based on the conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to MTT-formazan by mitochondrial enzyme.¹⁴ In brief, these cells were cultured with 1 ml of medium per well in 24-multiwell plates at 37°C for 24 h under 5% CO₂ in air. The cells were then treated with desired concentrations of urushiol. Then, the MTT solution (5 mg/mlPBS) was added to the well with a concentration of 0.25 mg/ml. Following the incubation for 4 h, the mixture of medium and the MTT solution was carefully discarded, after which 1 ml EtOH-DMSO (1 : 1) was added to the well in order to extract the crystallized dye. The amount of blue dye formed was determined by measuring the absorbance at 540 nm. Measurements were performed in triplicates.

After the cells were treated with or without half-maximal inhibition (IC₅₀) doses of urushiol (15 μg/ml for MKN-28 cells and 20 μg/ml for MKN-45 cells) for 48 h, they were washed twice with PBS containing 1% bovine serum albumin (PBS-B) and fixed with 70% ethanol containing 0.5% Tween 20 at 4°C for 30 min. Fixed cells were washed with PBS-B, and stained with propidium iodide (PI, Sigma-Aldrich) solution (50 μg/ml in PBS) for 30 min at room temperature. The stained cells were washed twice with PBS-B and observed via a fluorescence microscope at ×320 magnification.

Cells were harvested, rinsed twice in cold PBS, and resuspended in lysis buffer [5 mM Tris-HCl (pH 7.5), 5 mM ethylenediaminetetra acetic acid (EDTA), and 0.5% Triton X-100] at 4°C for 30 min. After centrifugating them at 27,000×g for 15 min, the supernatant was treated with RNase, followed by proteinase K digestion, phenol/chloroform/isoamyl alcohol (25 : 24 : 1) extraction and isopropanol precipitation. DNA separated through a 1.5% agarose gel was stained with ethidium bromide (EtBr, Sigma-Aldrich) and visualized by ultraviolet light source.

After the cells were treated with or without IC₅₀ doses of urushiol (15 μg/ml for MKN-28 cells and 20 μg/ml for MKN-45 cells) for 48 h, and washed with PBS, they were centrifuged (250×g for 10 min) and fixed as a pellet in 2.5% glutaraldehyde-1% osmium tetroxide buffered with PBS (pH 7.2), and processed for ultrathin sectioning by a slightly modified method.¹⁵ The samples were dehydrated in a graded ethanol series, embedded in Spury resin, and analyzed with standard procedures under a Hitachi H600-3 electron microscope (Tokyo, Japan).

The cells were harvested, lysed, and protein concentrations were quantified using the Bio-Rad protein assay (Bio-Rad Lab., Hercules, CA, USA) by the procedure described by the manufacturer. For the Western blot analysis, an equal amount of protein was subjected to electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA) by electroblotting. Blots were probed with the desired antibodies for 1 h, incubated with diluted enzyme-linked secondary antibodies and then visualized by the enhanced chemiluminescence (ECL) according to the recommended procedure (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The primary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Calbiochem (Cambridge, MA, USA). Peroxidase-labeled secondary antibodies were purchased from Amersham Pharmacia Biotech.

Data were expressed as the mean±SD of three separate experiments and analyzed by Student’s t-test. The means were considered significantly different at P<0.05.

The effect of urushiol on the viability of MKN-28 and MKN-45 cells was assessed via MTT dye assay at the concentrations of 3, 6, 12, and 24 μg/ml for 48 h. Urushiol showed cytotoxic and growth inhibitory effect on both cells in a concentration-dependent manner. The IC₅₀ values of urushiol were approximately 15 and 20 μg/ml on MKN-45 and MKN-28 cells, respectively (Fig. 1). Therefore, urushiol seemed to have stronger anti-proliferative activity on MKN-45 cells than MKN-28 cells.

There were significant morphological changes on MKN-45 and MKN-28 cells after the urushiol treatment (Fig. 2). However, the morphologies of two cell lines were significantly different. The sizes of MKN-45 cells were smaller than MKN-28 cells after urushiol treatment. The shape of MKN-45 cell was round, whereas MKN-28 cell was polygonal. Although apparent morphological changes were observed on urushiol-treated MKN-45 (Fig. 2A and B) and MKN-28 cells (Fig. 2C and D), any other differences between two cells were not detectable by light microscopy with no staining. To test whether decreased viability of human gastric cancer cells by urushiol was attributed to apoptosis, the structures of nucleus were studied with PI staining on urushiol-treated cells. In MKN-45 cells, morphological changes were corresponding to apoptosis, including nuclear fragmentation and condensation, observed by the treatment of 15 μg/ml urushiol (Fig. 2E and F). In contrast, any nuclear structural changes were not detectable on 20 μg/ml urushiol-treated MKN-28 cells (Fig. 2G and H).

To identify a hallmark of apoptotic cell death, assay for the genomic DNA fragmentation was performed. Urushiol treatment invoked DNA ladder formation, indicating that urushiol induced apoptosis in MKN-45 cell. The formation of DNA ladder was increased concentration-dependently (Fig. 3A). However, urushiol did not mediate DNA ladder formation on MKN-28 cells (Fig. 3B).

To confirm apoptotic cell death of MKN-45 by urushiol treatment, the cellular morphology was investigated via electron microscopy. The degeneration of apoptotic bodies observed on MKN-45 with the treatment of urushiol (Fig. 4A and B), whereas MKN-28 cells maintained intact cellular structure (Fig. 4C and D).

The p53 tumor suppressor gene is a cell cycle regulator and is able to induce cell cycle arrest to allow DNA repair or apoptosis.¹⁶ The product of p53 gene mediatesthe regulation of expression of apoptosis-related proteins and transactivates p21^WAF1/CIP1, CDK inhibitor.¹⁷ The treatment with urushiol mediated up-regulation of p53 and p21^WAF1/CIP1 expression on MKN-45 cell (Fig. 5). It was reported that Fas receptor and its ligand were involved in activation of apoptosis.¹⁸ Fas (48 kDa) and Fas-L (40 kDa) proteins were up-regulated on MKN-45 cells treated with urushiol (Fig. 5). Recently, it was reported that Bcl-2 family is a major regulator of apoptosis and Bcl-2/Bax ratio appears to be an important determinant in the regulation of apoptosis.¹⁹ To investigate the involvement of Bcl-2 family, the expression level of Bcl-2 and Bax was measured on MKN-45 and MKN-28 cells after treatment with urushiol. The expression level of Bcl-2 was very low and unchanged by urushiol on MKN-45 cells, whereas expressions of Bax were up-regulated by urushiol (Fig. 5). However, the expression of Bcl-2 was detectable without changing of expression by urushiol on MKN-28, whereas Bax expression was very low (data not shown).

Caspase-3 has known as executioner of apoptosis that mediated DNA fragmentation and cleavage of many cellular and nuclear proteins.²⁰ To study the involvement of caspase-3, the activation of caspase-3 and PARP cleavage were observed on MKN-45 cells treated with urushiol. After treatment of urushiol the level of proenzyme form (32 kDa) of caspase-3 was increased and active form (19 kDa) of enzyme was also detected on MKN-45 cells (Fig. 5). poly(ADP-ribose) polymerase (PARP), one of well-known substrates of caspase-3, was cleaved on MKN-45 cell by urushiol treatment (Fig. 5).

Although urushiol mediated apoptosis-associated changes on MKN-45 cell, it seemed that urushiol did not induce apoptosis on MKN-28 cells. DNA ladder formation and nuclear condensation were not observed after treatment with urushiol on MKN-28 cell (Fig. 3B, 4D). This implies that other mechanisms of growth inhibitory effect of urushiol on MKN-28 might be involved. To investigate the effects of urushiol on cell cycle, we studied expression of p21^WAF1/CIP1 protein, a well-known inhibitor of cyclin dependent kinase. The expression level of p21^WAF1/CIP1 protein was found to be increased by urushiol treatment on MKN-28 cell through a p53-independent manner (Fig. 6). Another inhibitor of cyclin dependent kinase, p27^KIP1, was also up-regulated by urushiol on MKN-28 cell (Fig. 6). However, cell cycle regulatory proteins at G1 phase, cyclin D1, was decreased by urushiol treatment on MKN-28 cell (Fig. 6). Additionally, cyclin dependent kinase 4 and cyclin dependent kinase 2 were slightly decreased on MKN-28 cell by urushiol treatment (Fig. 6).