Emodin Exerts an Antiapoptotic Effect on Human Chronic Myelocytic Leukemia K562 Cell Lines by Targeting the PTEN/PI3K-AKT Signaling Pathway and Deleting BCR-ABL

Abstract

The BCR-ABL kinase inhibitor, imatinib mesylate, is the front-line treatment for chronic myeloid leukemia, but the emergence of imatinib resistance has led to the search for alternative drug treatments. There is a pressing need, therefore, to develop and test novel drugs. Natural products including plants, microorganisms, and halobios provide rich resources for discovery of anticancer drugs. In this article, we demonstrate that emodin inhibited the growth of K562 cells harboring BCR-ABL in vitro and in vivo, and induced abundant apoptosis, which was correlated with the inhibition of PETN/PI3K/Akt level and deletion of BCR-ABL. These findings suggest that emodin is a promising agent to kill K562 cells harboring BCR-ABL.

Keywords: BCR-ABL; K562 cells; PI3K-AKT; PTEN; emodin.

Figure 1.

Effect of emodin on cell viability in K562 cells. (A) K562 cells were treated without or with emodin (25, 50, and 100 µmol/L for 24 hours). (B) K562 cells were treated without or with 50 µmol/L emodin for 24, 48, and 72 hours. Cell viability was evaluated with MTT assay as described in the Materials and Methods section. All results were expressed as mean ± SEM of 3 independent experiments (n = 3). *P < .05 and **P < .01 compared withcontrol.

Figure 2.

Cell cycle distributions of emodin-treated K562 cells were analyzed by flow cytometry. The cells were treated with 0.1% DMSO (negative control group), 25, 50, and 100 µmol/L emodin for 48 hours. (A) Representative data of flow cytometry assay. (B) All results were expressed as mean ± SEM of 3 independent experiments (n = 3). *P < .05 and **P < .01 compared withcontrol.

Figure 3.

Morphological changes induced by emodin in K562 cells. K562 cells were seeded in culture bottles and treated without (A and C) or with 50 µmol/L emodin (B and D) for 48 hours. Apoptotic cells (in B) were characterized by cellular shrinkage (light microscope, 100×). Under a transmission electron microscope, the apoptotic cell (in D) was characterized by marked condensation and margination of chromatin (5000×).

Figure 4.

The apoptotic rates of K562 cells induced by emodin. K562 cells were treated with 25, 50, and 100 µmol/L emodin for 24 hours. (A) Then cells were collected and stained with FITC-Annexin V to detect cell death phenomena by flow cytometry. Y axis shows PI-labeled population, and X-axis shows FITC-labeled Annexin-V positive cells. (B) The apoptotic rates of K562 cells induced by emodin. (*P < .05, **P < .01 vs control). All results were expressed as mean ± SEM of 3 independent experiments (n = 3). *P < .05 and **P < .01 compared withcontrol.

Figure 5.

The effect of emodin on the induction of apoptosis in K562 cells was determined using DNA fragmentation analysis. DNA was isolated from the cells and analyzed using 2% agarose gel electrophoresis with 1 × TBE buffer and ethidium bromide staining. The apoptotic characteristic laddering pattern of DNA fragments with multiple bands indicated the induction of apoptosis in emodin-treated cells at various conditions but not in the untreated control. Lane M: DNA ladder marker; Lane 1: 48 hours at 0 µmol/L (negative control); Lane 2: 48 hours at 25 µmol/L; Lane 3: 48 hours at 50 µmol/L; Lane 4: 48 hours at 100 µmol/L.

Figure 6.

The in vivoantitumor effect of emodin on athymic nude mice bearing K562 cells subcutaneously was studied. (A) The tumor size of xenografted tumor in nude mice with K562 cells in each group (control, emodin, and HU treatment groups). (B) Tumor volume of control, emodin, and HU treatment groups. Tumor volume was measured once every 2 days. (C) The weight of tumor of control, emodin, and HU treatment groups. After nude mice were treated for 12 days, the tumors were isolated and weighed. All results were expressed as mean ± SEM. *P < .05 and **P < .01 compared withcontrol.

Figure 7.

Emodin-induced histological changes of tumor mass in mice. BALB/c mice with established K562 cell tumors were injected with the indicated concentration emodin or HU one time every day and sacrificed for removing tumors. Tumor tissue was histological evaluated by hematoxylin-eosin staining. (A) negative control group; (B) emodin 25 mg/kg; (C) emodin 50 mg/kg; (D) emodin 100 mg/kg; (E) HU group. Sections were photographed under 200-fold (down) microscopy.

Figure 8.

Emodin-induced histological changes of tumor mass in mice. BALB/c mice with established K562 cell tumors were injected with the indicated concentration emodin or HU one time every day. Tumor tissue was histologically evaluated by electron microscope. Sections were photographed under indicated-fold transmission electron microscope. (A) Negative control group (×3500); (B) emodin 25 mg/kg (×5000); (C) emodin 50 mg/kg (×5000); (D) emodin 100 mg/kg (×5000); (E) HU 120 mg/kg (×5000).

Figure 9.

The mRNA expressions of PTEN, PI3K, AKT, and BCR-ABL in K562 cell from the emodin-treated group. RT-PCR analysis of the mRNA of PTEN, PI3K, AKT, and BCR-ABL obtained from K562 cell with the emodin or HU. M: DNA marker; 1: negative control; 2: 25 µmol/L group; 3: 50 µmol/L group; 4: 100 µmol/L group. All RT-PCRs were representative of 3 independent experiments (n = 3).

Figure 10.

The mRNA expressions of PTEN, PI3K, AKT, and BCR-ABL in K562 cell from the emodin-treated group. RT-PCR analysis of the mRNA of PTEN, PI3K, AKT, and BCR-ABL obtained from nude mice tumor xenografts treated with emodin or HU. M: DNA marker; 1: negative control; 2: 25 mg/kg group; 3: 50 mg/kg group; 4: 100 mg/kg group; 5: positive control HU group. All RT-PCR were representative of 3 independent experiments (n = 3).

Figure 11.

Involvement of PTEN, PI3K, AKT, and BCR-ABL proteins in emodin-induced apoptosis. (A) Western blotting analysis of PTEN, PI3K, AKT, and BCR-ABL proteins of K562 cells treated with the indicated concentration of emodin for 36 hours, respectively. (B) Western blotting analysis of PTEN, PI3K, AKT, and BCR-ABL of the protein extracts obtained from nude mice tumor xenograft treated with the indicated concentration of emodin or HU. All Western blots were representative of 3 independent experiments (n = 3).