Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c-myc, p53, and bcl-2 mRNA

Abstract

1. The mechanisms involved in the apoptotic effect of saikosaponin-d, a triterpene saponin from Bupleurum falcatum L., were studied in human CEM lymphocytes and compared with those of dexamethasone (3 x 10(-7) M). 2. Saikosaponin-d (10(-8) to 10(-5) M) inhibited the serum-stimulated [(3)H]-thymidine incorporation in a concentration-dependent manner. Dexamethasone also inhibited serum-stimulated [(3)H]-thymidine incorporation. 3. Cell viability was unaffected by saikosaponin-d until 10(-5) - 10(-4) M. Dexamethasone significantly reduced the number of viable cells. 4. Following saikosaponin-d (10(-5) - 10(-4) M) treatment, flow cytometry analysis of propidium iodide-stained cells showed a significant increase in the percentage of cells in the apoptotic region. Dexamethasone also significantly increased the percentage of apoptotic cells. The supravital exposure to propidium iodide and annexin V labelling demonstrated that saikosaponin-d (10(-5) - 10(-4) M) induced apoptosis as well as necrosis. 5. The apoptotic effect of saikosaponin-d (3 x 10(-6) - 10(-4) M) was also demonstrated by TUNEL analysis and DNA laddering. The percentage of apoptotic cells induced by saikosaponin-d (3 x 10(-6) - 10(-5) M) was unaffected by the presence of Z-VAD-FMK, indicating that saikosaponin-d-induced apoptosis may not be mediated by caspase activity. However, the percentage of apoptotic cells induced by dexamethasone was significantly reduced by the presence of Z-VAD-FMK. 6. Levels of c-myc, p53, and bcl-2 mRNA were analysed by the reverse transcription-polymerase chain reaction. Levels of c-myc and p53 mRNA were significantly increased, while the level of bcl-2 mRNA was decreased, by saikosaponin-d (10(-5) M) treatment. Dexamethasone did not significantly change the expression of these genes. 7. It is suggested that the apoptotic effect of saikosaponin-d may be partly mediated by increases in c-myc and p53 mRNA levels accompanied by a decrease in bcl-2 mRNA level.

Figure 1

Structure of saikosaponin-d.

Figure 2

Effects of saikosaponin-d on DNA synthesis in CEM cells. DNA synthesis was measured by uptake of [³H]-thymidine. The control value for serum-induced [³H]-thymidine incorporation in the absence of test compound was 9205±945 c.p.m. well⁻¹. The inhibitory activity of the test compound is expressed as a percentage of the control value (% of control). Each point with vertical line represents the mean and s.e. mean (n=5).

Figure 3

Effect of saikosaponin-d on CEM cell viability. Quiescent CEM cells (basal level, 2×10⁵ cells) were stimulated with serum. After addition of serum for 48 h in the absence (control level) or presence of test compound, the cells were harvested and their viabilities were examined by trypan blue dye exclusion test. The number of viable cells was estimated using a haemocytometer. Each column represents the mean±s.e.mean (n=5). *P<0.05, compared to the cell number in control group without saikosaponin-d and dexamethasone treatment (control level).

Figure 4

Effect of saikosaponin-d on the percentage of apoptotic CEM cells. Quiescent CEM cells were stimulated by serum in the absence or presence of test compound for the indicated time periods. The percentage of apoptotic cells untreated (control) and treated with saikosaponin-d for 12, 24 or 48 h was analysed by flow cytometric analysis of PI-stained cells as described in Methods. The effect of test compound was compared to that of 3×10⁻⁷ M dexamethasone. Each column represents the mean±s.e.mean (n=3 ∼ 6). #P<0.05, when compared to the control value (without test compound) for 12 h treatment. +P<0.05, when compared to the control value (without test compound) for 24 h treatment. ^*P<0.05, when compared to the control value (without test compound) for 48 h treatment.

Figure 5

Contour diagram of annexin V/PI flow cytometry of CEM cells. CEM cells untreated (a), or treated with saikosaponin-d, 3×10⁻⁶ M (b) or 10⁻⁵ M (c), or 3×10⁻⁷ M dexamethasone (d) were performed PI and annexin V labelling as described in the Methods. The lower left quadrant of each panel (LL, annexin V⁻PI⁻) shows the viable cells, which exclude PI and are negative for annexin V binding. The lower right quadrant (LR, annexin V⁺PI⁻) represents the early apoptotic cells, annexin V positive and PI negative, demonstrating cytoplasmic membrane integrity. The upper right quadrant (UR, annexin V⁺PI⁺) contains advanced apoptotic cells and necrotic cells, which are positive for annexin V binding and for PI uptake. The percentages of cells in these three quadrants were calculated from four experiments and were summarized as shown in the table.

Figure 6

TUNEL analysis of CEM cells. CEM cells treated with (a) 10⁻⁶ M saikosaponin-d, (b) 10⁻⁵ M saikosaponin-d, (c) 10⁻⁴ M saikosaponin-d, (d) control, untreated with test compound, or (e) treated with 3×10⁻⁷ M dexamethasone were taken through the TUNEL procedure. TUNEL-positive cells were visualized using a peroxidase-substrate system as having brown nuclei. The green-blue nuclei as counter-stained with methyl green indicate TUNEL-negative. Few nuclei in untreated cells were positively labelled for DNA fragmentation, while a large proportion of the nuclei in 10⁻⁵ and 10⁻⁴ M saikosaponin-d-treated cells or 3×10⁻⁷ M dexamethasone-treated cells were TUNEL-positive. Bar=12 μm. Similar results were obtained in three independent experiments.

Figure 7

Electrophoresis of fragmented DNA in CEM cells. Genomic DNA was isolated from untreated (control) cells and cells treated with saikosaponin-d or dexamethasone for 48 h. DNA fragmentation was evaluated by electrophoresis on agarose gel containing ethidium bromide and photographed under UV light. The DNA ladder was detected in cells treated with 10⁻⁵ or 10⁻⁴ M saikosaponin-d. The effect of test compound was compared to that of 3×10⁻⁷ M dexamethasone. Lane M, ΦX174/HaeIII DNA size marker; lane 1, untreated control; lane 2, treated with 10⁻⁶ M saikosaponin-d; lane 3, treated with 10⁻⁵ M saikosaponin-d; lane 4, treated with 10⁻⁴ M saikosaponin-d; lane 5, treated with 3×10⁻⁷ M dexamethasone. Similar results were obtained in three independent experiments.

Figure 8

Effects of saikosaponin-d on c-myc, p53, and bcl-2 mRNA levels in CEM cells. Quiescent CEM cells were stimulated by serum in the absence or presence of test compound for 12 h. The serum-stimulated test gene mRNA levels in cells untreated or treated with either 10⁻⁵ M saikosaponin-d or 3×10⁻⁷ M dexamethasone were analysed by RT–PCR amplification as described in the Methods. Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The signal intensities of test genes and GAPDH were quantified using image analyser, and the changes in the signal intensities of the test genes relative to GAPDH were calculated. Results are expressed as percentages of the control level without test compound (% of control) (bottom of figure) (n=3); the corresponding electrophoretic patterns of PCR products are shown at the top of each panel. Lane M, Φ X174/HaeIII DNA size marker; lane 1, 4, 7, control without test compound; lane 2, 5, 8, 10⁻⁵ M saikosaponin-d-treated; lane 3, 6, 9, 3×10⁻⁷ M dexamethasone-treated. c-myc mRNA level: lane 1, 2, 3; p53 mRNA level: lane 4, 5, 6; bcl-2 mRNA level: lane 7, 8, 9; lane N, negative control, no RT. Each column represents the mean±s.e.mean (n=3). *P<0.05, when compared to the control value without test compound.