Human gastric adenocarcinoma cell line SGC-7901, obtained from Wuhan University Type Culture Collection (Wuhan, China), was routinely maintained in phenol red-free RPMI1640 (Gibco BRL, Grand Island, NY) containing 100 mL/L fetal bovine serum (Hyclone, Logan, UT), 10⁵ U/L penicillin and 100 mg/L streptomycin at 37 °C in a humidified atmosphere with 50 mL/L CO₂ in air. When cells were grown to approximately 50% confluence, medium was replaced with serum-free RPMI1640. After 24 h, fresh media containing 2.5, 5, 10, 20 μmol/L mifepristone (Sigma Chemical Co., St Louis, MO) were added, respectively. Control cells were treated with the same volumes of vehicle (ethanol). Unless otherwise indicated, the cells were harvested after 96 h of incubation.

The expression of PR mRNA in SGC-7901 cells was detected by in situ hybridization (ISH) using an ISH detection kit for PR(Boster, Wuhan, China) according to the manufacturer’s instructions. Unless otherwise stated, all steps were performed at room temperature. Briefly, after 24 h of culture on the RNAse-free slides, cells were washed 3 times with phosphate buffered saline (PBS, pH7.4), fixed with 40 g/L paraformaldehyde in PBS containing 0.1 g/L diethylpyrocarbonate (DEPC-water) for 30 min, washed 3 times with 0.01 mol/L PBS, and then incubated with 5 mL/L hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. After being rinsed with 0.01 mol/L PBS, cells were digested with proteinase K (10 g/mL in 0.01 mol/L PBS) at 37 °C for 15 min. Further washes with 0.5 mol/L PBS were performed before pre-hybridization for 3 h at 37 °C in the pre-hybridization solutions in a humidified environment. Hybridization was then performed with digoxigenin-labeled cRNA antisense probe (5 g/mL) overnight at 37 °C in a moist chamber. Subsequently cells were washed for 10 min with 2 SSC (1 SSC = 150 mmol/L NaCl, 15 mmol/L sodium citrate, pH7.0), followed by 0.5 SSC for 15 min, and finally 0.2 SSC for 15 min. After treatment with blocking reagent for 30 min, cells were incubated with biotin-labeled mouse anti-digoxigenin antibody at 37 °C for 1 h, washed 4 times, for 5 min each time, with 0.5 mol/L PBS, and then treated with SABC solutions at 37 °C for 20 min. Then cells were washed 3 times, for 5 min each time, with 0.5 mol/L PBS, incubated with biotin-labeled peroxidase (POD) at 37 °C for 20 min, washed three time with 0.5 mol/L PBS. Finally, cells were visualized with 3,3-diaminobezidine (DAB), counterstained with hematoxylin, dehydrated, cleared, mounted with neutralgum, and examined under an microscope. Brown-yellow deposits indicated the sites of hybridization. PR-positive breast cancer tissues were used as positive control, and probes were replaced by PBS as negative control.

Harvested cells were washed 3 times with PBS, fixed for 2 h with 2.5 g/L glutaraldehyde in PBS, and then post-fixed for 2 h at 4 °C with 1 g/L OsO₄ in PBS. Cells were dehydrated using gradually increasing concentrations of ethanol from 50% to 100%, and then embedded in Epon 812. The ultra-thin sections (60 nm) were stained with uranyl acetate and lead citrate prior to examination at 50 kV with a Hitachi 600 transmission electron microscrope (Hitachi Corp., Tokyo, Japan).

SGC-7901 cells were seeded into 96-well plates at a density of 1 × 10⁵/mL in RPMI1640. After 96 h of incubation with various concentrations of mifepristone, cell proliferation was measured by MTT (Sigma) reduction assay as described previously[15]. Absorbance at 570 nm (A_570nm) was assayed. The inhibitory rate (IR) of SGC-7901 cells was calculated according to the equation as following: IR (%) = (A_570nm in control group - A_570nm in mifepristone-treated group)/A_570nm in control group × 100%.

Cells were incubated at various time intervals without or with various concentrations of mifepristone, followed by treatment with 10 μCi ³H-TdR (Amersham, Arlington Heights, IL) for an additional 6 h. Then, cells were washed twice with 100 mL/L trichloroacetic acid by centrifugation and resuspension, and were continuously incubated for 30 min at 60 °C with 0.5 mL of NaOH (0.3 mol/L). Finally, the cell lysates were collected, and the radioactivity was measured by a liquid scintillation counter(Beckman LS1 801, USA).

The harvested cells were fixed with 700 mL/L ethanol at -20 °C for 30 min, and then stained with propidium iodide (Sigma) for 30 min in the dark. The stained cells were analyzed in a FACS Calibur flow cytometer (Becton Dickinson Labware, Lincoln Park, NJ) with excitation wavelength of 488 nm. The resulting histograms were analyzed by program MODFIT for cell distribution in cell cycle phase. Proliferative index (PI) was calculated according to the formula: PI (%) = (S + G₂/M)/(G₀/G₁ + S + G₂/M) × 100%.

Total proteins were extracted from harvested cells as described previously[16], and protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). An equal amount of cellular protein from extract of each group was added to a final volume of 100 μL of reaction mixture containing 0.2 mmol/L of a colorimetric caspase-3 substrate, acetyl-Asp-Glu-Val-Asp-ρ-nitroaniline (Ac-DEVD-ρNA; Calbiochem, San Diego, CA), followed by incubation at 30 °C for 10 min. Free ρ-nitroaniline (ρNA) released upon enzymatic cleavage was detected at 405 nm using a microplate reader (Bio-Rad). Caspase-3 activity correlated with the concentration of free ρNA generated in the reaction. Purified caspase-3 (Calbiochem) was used as positive control, whereas caspase-3 inhibitor I (Ac-DEVD-CHO, Calbiochem) was used as negative control.

Total RNA was extracted from the cells using TRIzol reagent(Gibco BRL) according to the manufacturer’s protocol. Two milligrams of total RNA were used for reverse transcription in a total volume of 20 μL with the SuperScript preamplification system (Promega, Madison, MI). Aliquots of 2 μL cDNA were subsequently amplified in a total volume of 50 μL using the GeneAmp PCR kit (Promega) following conditions recommended by the manufacturer. The sense and antisense primers for Bcl-X_L were 5’-AGGCAGGCGATGAGTTTGAAC-3’ and 5’-GAACCACACCAGCCACAGTCA-3’, respectively. The sense and antisense primers for -actin used as an internal control were 5’-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3’ and 5’-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3’, respectively. The cycling conditions were 94 °C for 2 min, followed by 30 cycles of 92 °C for 30 s, at 62 °C for 30 s, and at 72 °C for 1 min and a final extension of 72 °C for 5 min. PCR products were separated on the 15 g/L agarose gel stained with ethidium bromide (EB) and viewed under ultraviolet light.

Data were expressed as mean ± SD. Statistical analysis was performed using the Student’s t test and the chi-square test. P < 0.05 was considered statistically significant.

In situ hybridization analysis showed that PR mRNA was highly expressed in the cultured SGC-7901 cells, which was mainly localized in the cytoplasm of the cells (Figure 1).

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Figure 1 In situ hybridization (ISH) analysis of progesterone receptor (PR) expression in human gastric adenocarcinoma cell line SGC7901 (ISH, × 1000).

To assess the effect of mifepristone on the ultrastructural changes of SGC-7901 cells, transmission electron microscopic analysis was performed. Results revealed that mifepristone dose-dependently induced apoptosis, which was especially remarkable at the 20 μmol/L concentration (Figure 2B). However, the irregular and enlarged nuclear, multiple nucleoli and increased nucleus-to-cytoplasm ratio were clearly seen in the cells of control group (Figure 2A).

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Figure 2 Transmission electron microscopic photographs of the SGC7901 cells cultured for 96 h in the absence (A) or the presence of 20 μmol/L mifepristone (B) in vitro (TEM, × 2000). Arrows indicate apoptotic bodies which were formed in the cells of mifepristone-treated group.

After 96 h of incubation with 2.5, 5, 10, 20 μmol/L mifepristone, MTT assay revealed A_570nm was markedly decreased in a dose-dependent manner, and the inhibitory rate (IR) of SGC-7901 cells by mifepristone was 8.98%, 20.36%, 39.73% and 51.29%, respectively (Table 1). Figure 3 shows that ³H-TdR incorporation into DNA of SGC-7901 cells was significantly decreased in a dose- and time-dependent manner.

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Figure 3 Effect of various concentrations of mifepristone on the ³H-TdR incorporation of SGC7901 cells at various time intervals in vitro.

The effect of mifepristone on the cell-cycle phase distribution of SGC-7901 cells was determined by flow cytometry. After treatment with mifepristone, there was a strong dose- dependent decrease in the percentage of S- and G₂/M-phase cells, and with a concomitant increase in the percentage of cells in the G₀/G₁ phases of the cell cycle (Table 1). Additionally, there is a significant decrease in the proliferative index (PI) of the mifepristone-treated cells (50.53%, 47.69%, 34.20% and 22.83%) as compared with control group (57.75%, P < 0.01).

As shown in Table 1, mifepristone significantly up-regulated the activity of caspase-3 as compared with that in control group. Figure 4 shows the results of RT-PCR analysis for Bcl-x_L mRNA expression. Results indicated that mifepristone dose-dependently inhibited the expression of Bcl-X_L in the SGC-7901 cells.

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Figure 4 RT-PCR analysis of Bcl-X_L mRNA expression in the SGC7901 cells cultured for 96 h in the absence or the presence of various concentrations of mifepristone in vitro. Lanes 1-5: Marker (bp), contro l, 5, 10, 20 μmol/L mifepristone, respectively.