Induction of Apoptosis Scutellaria baicalensis Georgi Root Extract by Inactivation of the Phosphatidyl Inositol 3-kinase/Akt Signaling Pathway in Human Leukemia U937 Cells

Abstract

Background: The roots of Scutellaria baicalensis Georgi (Labiatae) have been widely used in traditional medicine for treatment of various diseases. In this study, we investigated the effects of ethanol extracts of S. baicalensis roots (EESB) on the growth ofn human leukemia U937 cells.

Methods: The effect of EESB on cell viability was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was determined using 4,6-diamidino-2-phenyllindile staining and flow cytometry. The effects of EESB on the expression of regulatory proteins of apoptosis and phosphatidyl inositol 3-kinase (PI3K)/Akt signaling were determined by Western blotting. Caspase activity and mitochondrial membrane potential (MMP) were measured using flow cytometric analysis.

Results: EESB significantly inhibited the growth of U937 cells and induced apoptosis, which was associated with down-regulation of anti-apoptotic Bcl-2, up-regulation of pro-apoptotic Bax, the loss of MMP and activation of caspase-9 and -3. We also found that EESB enhanced the expression of death receptors (DRs) and their associated ligands and induced the activation of caspase-8 and truncation of Bid. In addition, EESB suppressed PI3K/Akt signaling and EESB-induced apoptosis and growth inhibition were further increased by inhibition of PI3K activity.

Conclusions: Our results indicated that the pro-apoptotic effect of EESB was mediated through the activation of DR-mediated intrinsic and mitochondria-mediated extrinsic apoptosis pathways and inhibition of the PI3K/Akt signaling in U937 cells.

Keywords: Apoptosis; Caspase; PI3K/Akt; Scutellaria baicalensis; U937 cells.

Figure 1

Effects of ethanol extracts of Scutellaria baicalensis roots (EESB) on cell viability in various cancer cell lines. Hep3B hepatoma (A), B16F10 melanoma (B), U937 leukemia (C), MDA-MB-231 and MCF-7 breast carcinoma (D, E), and A549 lung carcinoma (F) cells were treated with the indicated concentrations of EESB for 24 hours. The cell viabilities were determined by the MTT assay. Data are presented as mean ± SD values obtained from three independent experiments. *P < 0.05 compared with the untreated control group.

Figure 2

Induction of apoptosis by ethanol extracts of Scutellaria baicalensis roots (EESB) in U937 cells. Cells were plated onto 6-well plates at a density of 4.0 × 10⁵ cells/well and treated with variable concentrations of EESB for 24 hours. (A) Nuclei stained with 4,6-Diamidino-2-phenylindole solution were photographed with a fluorescence microscope using a blue filter (×400). Arrows indicate apoptotic cells. (B, C) To quantify the degree of cell apoptosis, the cells were stained with annexin V-FITC and propidium iodide (PI), and the percentage of apoptotic cells (annexin V⁺ cells) was determined using flow cytometric analysis. Data are presented as mean ± SD values obtained from three independent experiments. *P < 0.05 compared with the untreated control group.

Figure 3

Effects of ethanol extracts of Scutellaria baicalensis roots (EESB) on the expression of death receptor (DR)-related and inhibitor of apoptosis protein (IAP) proteins in U937 cells. Cells grown under the same conditions (shown in Figure 2) were lysed, and cellular proteins were visualized using the indicated antibodies and an enhanced chemiluminescence detection system. Actin was used as an internal control.

Figure 4

Activation of caspases and degradation of caspase substrates by ethanol extracts of Scutellaria baicalensis roots (EESB) in U937 cells. (A) Cells grown under the same conditions (shown in Figure 2) were lysed, and cellular proteins were visualized using the indicated antibodies and an enhanced chemiluminescence detection system. Actin was used as an internal control. (B) Cells were lysed, and aliquots (50 μg protein) were assayed for in vitro caspase-3, -8, and -9 activity using DEVD-p-nitroaniline (pNA), IETD-pNA, and LEHD-pNA as substrates, respectively, at 37°C for 1 hour. The released fluorescent products were analyzed. Data are presented as mean ± SD values obtained from three independent experiments. *P < 0.05 compared with the untreated control group.

Figure 5

Effects of ethanol extracts of Scutellaria baicalensis roots (EESB) on the expression of Bcl-2 family proteins and values of mitochondrial membrane potential (MMP) in U937 cells. (A) Cells were treated with EESB for 24 hours, and aliquots containing the total protein levels were subjected to SDS PAGE followed by immunoblot analysis with the specific antibodies. (B, C) To quantify the degree of MMP loss, untreated control and EESB-treated cells were incubated with 10 μM JC-1 for 20 minutes at 37°C in the dark. The cells were then washed once with PBS, and MMP loss was evaluated using a flow cytometer. Data are presented as the mean ± SD values obtained from three independent experiments. *P < 0.05 compared with the untreated control group.

Figure 6

Inactivation of the phosphatidyl inositol 3-kinase (PI3K)/Akt signaling pathway by ethanol extracts of Scutellaria baicalensis roots (EESB) in U937 cells. The cells were treated with EESB (200 mg/ml) for the indicated times. Equal amounts of cell lysate were resolved on SDS PAGE, transferred to nitrocellulose membranes and probed with the anti-p-PI3K, anti-PI3K, anti-p-Akt and anti-Akt antibodies. The proteins were visualized using an enhanced chemiluminescence detection system. Actin was used as an internal control.

Figure 7

Increase in ethanol extracts of Scutellaria baicalensis roots (EESB)-induced apoptosis by inactivation of phosphatidyl inositol 3-kinase/Akt signaling in U937 cells. The cells were pre-treated with or without LY294002 (50 mM) for 1 hour before treatment with EESB (200 mM) for 24 hours. (A, B) Mitochondrial membrane potential (MMP) loss was measured using a flow cytometer. (C) Nuclei stained with 4,6-Diamidino-2-phenylindole solution were photographed with a fluorescence microscope using a blue filter (×400). Arrows indicate apoptotic cells. (D) The percentage of apoptotic cells (annexin V⁺ cells) was analyzed using a flow cytometer. (E) Cell viability was determined by an MTT assay. Each point represents the mean ± SD value of three independent experiments. *P < 0.05 vs. untreated control cells.