Keratinocytes of the naturally immortalized normal cell line, HaCaT, were obtained from the Laboratory of Apoptosis, College of Life Science, Hunan Normal University, China. The human embryonic lung fibroblast cell line (human normal fibroblast cell line), Hel, was established by the Laboratory of Molecular Virology, College of Life Science, Hunan Normal University, China (14).

The impact of arecoline treatment on cell proliferation was measured by the MTT assay as described previously (15). Briefly, keratinocytes from the natural immortalized normal cells, HaCaT, and human embryonic lung fibroblast cells, Hel, (10⁴ cells/well) were cultured in triplicate with 10% FCS RPMI-1640 in 96-well plates in the presence or absence of 25, 50, 75, 100 and 125 μg/ml arecoline for 0, 24, 48 and 72 h, respectively. The cells were then exposed to 5 mg/ml MTT for 4 h. The generated formazan was dissolved with dimethyl sulfoxide, and the absorbance was measured at 570 nm using an ELX-800 microplate reader (Bio Tek Instruments, Inc., Winooski, VT, USA).

The previously described keratinocytes and lung fibroblast cells were cultured in 10% FCS RPMI-1640 to ~70% confluence and then treated with, or without, 25, 50, 75, 100 and 125 μg/ml arecoline for 12 h, respectively. The adherent cells were trypsinized, harvested and fixed in 70% ethanol at 4°C. Subsequently, the cells were washed with cold PBS and stained with propidium iodide (PI) in working solution (0.5 mg/ml RNase, and 0.1 mg/ml PI in PBS). The cell cycle was characterized by flow cytometric analysis using a FACScan cytofluorimeter, and the data were analyzed by the CellQuest Pro Software (Becton-Dickinson, San Jose, CA. USA).

Both cell lines were cultured in RPMI-1640 to 80% confluence and then treated with, or without, 25, 50, 75, 100 and 125 μg/ml arecoline for 12 h, respectively. The cells were harvested, and their total RNA was extracted. One microgram total RNA was reversely transcripted into cDNA, as described previously (15,16) and was subsequently used as the template for PCR reaction. The first step in the PCR reaction involved denaturing at 94°C for 5 min, followed by 30 cycles of 94°C for 45 sec, 55°C for 45 sec, and 72°C for 1 min, followed by 72°C for 7 min for CDK2, CDK4, cyclin A, cyclin B, cyclin D1, cyclin E, E2F1 and CDC2. The primers CDK2, CDK4, cyclin A, cyclin B, cyclin D1, cyclin E, E2F1, CDC2 and β-actin were synthesized as shown in Table I. All RT-PCR reactions were repeated at least three times with varying cycle numbers to avoid potentially false results. β-actin was used as an endogenous control for normalization. The PCR products were analyzed by agarose-gel electrophoresis and ethidium bromide staining.

The ketatinocytes and the epithelial cells were cultured in RPMI-1640 up to 80% confluence and then treated with, or without, 25, 50, 75, 100 and 125 μg/ml arecoline for 12 h, respectively. The cells were harvested and lysed in the lysis buffer [1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 50 mM NaF, 2 mM EDTA, 400 mM NaCl, 10% glycerol plus complete protease inhibitor mixture (Roche Diagnostics)]. The protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA). The cell lysates (50 μg) from individual samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (HyClone Laboratories, Logan, UT, USA). The membranes were blocked with 5% nonfat milk in Tris-buffered saline/Tween-20 (25 mM Tris-HCl, 150 mM NaCl, pH 7.5, and 0.05% Tween-20) and probed with the primary antibody overnight at 4°C. After washing with Tris-buffered saline/Tween-20 three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and the specific signals were visualized using ECL detection system. Antibodies against cyclin D1, CDK4, CDK2, E2F1, cyclin B, CDC2, cyclin A and cyclin E were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Differences in nonparametric variables were analyzed by the Fisher's exact test using the EPI software (EPI Info, version 3.2.2, www.CDC.gov/epiinfo/). Differences in the quantitative variables between groups were analyzed by the Student's t-test using SPSS 11.0 program (SPSS, Chicago, IL, USA). A P-value <0.05 was considered to indicate a statistically significant result.

The HaCaT cell morphology was significantly altered following treatment of arecoline at different concentrations after 48 h. The outlines of the HaCaT cells were clear when treated with 0 and 25 μg/ml, but when the concentration of arecoline increased to 50 μg/ml, the boundaries of the HaCaT cells became indistinct and some of the cells were dead. There was massive cell death and a lack of boundaries between the cells when the concentration of arecoline was increased to 75, 100 and 125 μg/ml (Fig. 1). In contrast, the Hel cells were less affected by arecoline treatment. Only when the concentration of arecoline was increased to 100 and 125 μg/ml, did the Hel cell morphology begin to change slightly (Fig. 2).

To further test whether arecoline modulates HaCaT cell growth, HaCaT and Hel cells were treated with 0, 25, 50, 75, 100 and 125 μg/ml arecoline, and their spontaneous proliferation was determined by MTT assays. Treatment with arecoline significantly inhibited the proliferation of HaCaT cells (Fig. 3A), but only slightly reduced Hel cell growth in vitro (Fig. 3B). Therefore, treatment with arecoline inhibited the proliferation of epithelial cells, but not fibroblasts in vitro.

Inhibition of cell proliferation usually is mediated by inducing cell cycle arrest. To test this possibility, HaCaT and Hel cells were treated with 0, 25, 50, 75, 100 and 125 μg/ml arecoline and the cell cycle distribution was determined by FACS analysis (Tables II and III). In comparison to the control cells without arecoline treatment, following treatment with ≥75 μg/ml arecoline a higher percentage of HaCaT cells remained at the G₀/G₁ phase of the cell cycle (from 49.02 to 73.00%) at 12 h, accompanied by a reduced percentage of cells in the S phase (from 32.05 to 20.50%). However, arecoline treatment did not significantly alter Hel cell cycling even after treatment with arecoline for 12 h. Hence, these data demonstrate that arecoline treatment induces cell cycle arrest by inhibiting the G₁ to S phase transition of the cell cycle in epithelial cells in vitro.

To reveal the possible mechanism of the effect of arecoline on epithelial cells in vitro, the levels of mRNA transcripts of several cell cycle regulatory molecules (CDK2, CDK4, cyclin A, cyclin B, cyclin D1, cyclin E, E2F1, CDC2) were determined by semi-quantitative RT-PCR. Cyclin D1 mRNA expression was downregulated following arecoline stimulation, while there was no significant concentration gradient dependence. The mRNA expression of cyclin B was upregulated. Low concentrations of arecoline stimulated CDK4 expression. However, its expression was suppressed following treatment with high concentrations of arecoline. Cyclin A expression level was increased in the HaCaT cells following treatment with arecoline. Cyclin E expression level was decreased with an increase in arecoline concentrations. CDK2 had the same tendency as cyclin E. There was no significant difference in CDC2 levels (Fig. 4).

To confirm the change mechanism of cell cycle regulatory molecules, we tested the protein levels of CDK2, CDK4, cyclin A, cyclin B, cyclin D1, cyclin E, E2F1 and CDC2 by western blotting. The expression levels of cyclin D1, CDK4, CDK2 and E2F1 were significantly downregulated. Expression of cyclin A and cyclin E was slightly upregulated in HaCaT cells following arecoline stimulation. There was no significant difference in cyclin B and CDC2 expression (Fig. 5).