Emodin Induces Apoptotic Death in Murine Myelomonocytic Leukemia WEHI-3 Cells In Vitro and Enhances Phagocytosis in Leukemia Mice In Vivo

Abstract

Emodin is one of major compounds in rhubarb (Rheum palmatum L.), a plant used as herbal medicine in Chinese population. Although many reports have shown that emodin exhibits anticancer activity in many tumor cell types, there is no available information addressing emodin-affected apoptotic responses in the murine leukemia cell line (WEHI-3) and modulation of the immune response in leukemia mice. We investigated that emodin induced cytotoxic effects in vitro and affected WEHI-3 cells in vivo. This study showed that emodin decreased viability and induced DNA fragmentation in WEHI-3 cells. Cells after exposure to emodin for 24 h have shown chromatin condensation and DNA damage. Emodin stimulated the productions of ROS and Ca(2+) and reduced the level of ΔΨ(m) by flow cytometry. Our results from Western blotting suggest that emodin triggered apoptosis of WEHI-3 cells through the endoplasmic reticulum (ER) stress, caspase cascade-dependent and -independent mitochondrial pathways. In in vivo study, emodin enhanced the levels of B cells and monocytes, and it also reduced the weights of liver and spleen compared with leukemia mice. Emodin promoted phagocytic activity by monocytes and macrophages in comparison to the leukemia mice group. In conclusions, emodin induced apoptotic death in murine leukemia WEHI-3 cells and enhanced phagocytosis in the leukemia animal model.

Figure 1

Emodin affected the percentage of viable cells and DNA fragmentation in WEHI-3 cells. Cells (1 × 10⁴ cells/well; 96-well plates) were plated in RPMI 1640 medium + 10% fetal bovine serum (FBS) with 0, 25, 50, 100, and 150 μM of emodin for 24 and 48 h. The cells were collected by centrifugation and the viable cells were determined by using the MTT assay (a). Cells were treated with 0, 50, and 100 μM of emodin for 24 h, and then DNA was isolated for DNA gel electrophoresis (b) as described in Section 2. Columns, mean of three determinations; bars, SD. a, P < .05 shows significantly different when compared with DMSO-treated control; b, c, and d, P < .05 indicates significantly different compared with 25, 50, and 100 μM emodin-treated groups, respectively (one-way ANOVA followed by Bonferroni's test for multiple comparisons).

Figure 2

Effects of emodin on apoptosis and DNA damage in WEHI-3 cells by using DAPI staining and Comet assay. Cells (2 × 10⁵ cells/well) in 12-well plate were incubated with 0, 25, 50, and 100 μM emodin for 24 h and apoptosis was determined using DAPI staining (a). Data represent mean ± SD of at least three experiments. Cells were treated with 0, 50, and 100 μM emodin for 24 h and then were harvested for the examination of DNA damage using the Comet assay (b) as described in Section 2. Comet tail length was calculated, quantified and expressed in mean ± S.D for at least three replicates. *Significantly different compared with DMSO-treated control, P < .05, and ***significantly different from the control sample at P < .001.

Figure 3

Emodin affected the levels of ROS, Ca²⁺ and ΔΨ_m in WEHI-3 cells. Cells were cultured in 100 μM emodin for 0, 1, 3, 6, 12 or 24 h. Cells were harvested and resuspended in DCFH-DA for determining the changes in ROS (a), in Fluo-3/AM for staining of Ca²⁺ (b) and in DiOC₆ for determining ΔΨ_m (c) as described in Section 2. *P < .05 and ***P < .001 were considered significant when compared with DMSO-treated control.

Figure 4

Emodin stimulated the activities of caspase-3 and -9 of WEHI-3 cells. Cells were seeded in RPMI 1640 medium + 10% FBS with pretreatment with NAC, inhibitors of caspase-9 and -3 or a pan-caspase inhibitor, and then were exposed to 100 μM of emodin for 24 h. Cells were determined the caspase-3, -8, and -9 activity (a) and percentage of viable cells (b) as described in Section 2. Each experiment was done with triple sets, and columns, mean of three determinations; bars, SD. *P < .05 and ***P < .001 were considered significant different as compared to the DMSO-treated control group.

Figure 5

Emodin affected the apoptosis-associated protein levels in WEHI-3 cells. Cells were treated with emodin at 100 μM for 0, 6, 12, and 24 h, and then the total proteins were prepared and determined as described in Section 2. The levels of associated proteins expressions ((a): caspase-3, -7, and -9 and PARP; (b): cytochrome c, Apaf-1, AIF and Endo G; (c): GADD153, GRP78, ATF-6α and caspase-12; (d): Bcl-2, Bcl-xL, Bax, and Bad) were estimated by Western blotting as described in Materials and Methods.

Figure 6

The proposed mechanisms of emodin-induced apoptosis in WEHI-3 cells. The flow chart shows that emodin induced apoptosis through the ER stress, mitochondria-, and caspase-3-dependent signaling pathways in murine leukemia WEHI-3 cells in vitro.

Figure 7

Emodin affected the weights of the leukemia mice which were treated with without or with emodin 2 weeks. BALB/c mice were intraperitoneally injected with WEHI-3 cells (1 × 10⁵ cells/100 μL) in PBS for 2 weeks and/or treated with emodin once daily by oral administration for 14 days. Blood was collected and animals were sacrificed for examinations of weights of body (a) spleen (b) and liver (c) tissues, and then were individually weighed. Each point is the mean ± SD and similar results were observed in at least three independent experiments (n = 10) followed by one-way ANOVA followed by Dunnett's test.

Figure 8

Emodin affected the cell markers of white blood cells from leukemia BALB/c mice. The leukemia mice were orally treated without and with emodin (5 and 10 mg/kg) in olive oil for 14 days. Blood was collected from individual animals and was analyzed for surface cell markers by flow cytometry as described in Section 2. Each point is the mean ± SD and similar results were observed in at least three independent samples (n = 10) followed by one-way ANOVA followed by Dunnett's test. N.S. = Not Significant (P > .05).

Figure 9

Emodin affected on phagocytosis by monocytes and macrophages from leukemia BALB/c mice. Mice were injected with WEHI-3 cells (1 × 10⁵ cells/100 μL) in PBS for 2 weeks and treated without or with emodin for 2 weeks. Leukocytes were collected from PBMC (a) and peritoneal cavity (b) from animals and were analyzed for phagocytosis by flow cytometry as described in Section 2. Each point is the mean ± SD and similar results were observed in at least three independent samples (n = 10), and *P < .05 and ***P < .001 (n = 10) were shown significant followed by one-way ANOVA followed by Dunnett's test.