Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine

Abstract

Objectives: Meconopsis integrifolia (M. integrifolia) is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects.

Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS) level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP) loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C.

Results: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis.

Conclusions: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

Keywords: G2/M phase arrest; K562 cells; M. integrifolia ethanol extract; apoptosis; mitochondria.

Figure 1

Cell viability tests. (A) K562 cells viability after M. integrifolia treatment was measured by MTT assay; (B) Cytotoxicity effect of M. integrifolia on PBMCs and K562 cells through Typan blue assay; (C) After treatment with different concentrations of M. integrifolia for 24, 48 and 72 h, cell morphology was observed by a phase-contrast microscopy. Each value was expressed as a mean ± S.D. of at least three independent determinations. One-way ANOVA was used for comparisons of multiple group means followed by Dunnett’s t-test. ** p < 0.01 vs. Control. (Error bars = S.D., n = 3).

Figure 2

DNA damage and cell cycle arrest post M. integrifolia treatment. (A) Effect of M. integrifolia on DNA fragmentation of K562 cells. K562 cells were treated with M. integrifolia for 24, 48 and 72 h, then stained with propidium iodide (PI) and analyzed by flow cytometry; (B) Cell cycle analysis of M. integrifolia-treated cells. K562 cells were harvested and fixed in 70% alcohol and then stained with PI. Finally the stained cells were analyzed using a flow cytometer. ** p < 0.01 vs. Control. (Error bars = S.D., n = 3).

Figure 2

DNA damage and cell cycle arrest post M. integrifolia treatment. (A) Effect of M. integrifolia on DNA fragmentation of K562 cells. K562 cells were treated with M. integrifolia for 24, 48 and 72 h, then stained with propidium iodide (PI) and analyzed by flow cytometry; (B) Cell cycle analysis of M. integrifolia-treated cells. K562 cells were harvested and fixed in 70% alcohol and then stained with PI. Finally the stained cells were analyzed using a flow cytometer. ** p < 0.01 vs. Control. (Error bars = S.D., n = 3).

Figure 3

Apoptosis detection in M. integrifolia-treated cells. (A) The quantification of apoptotic cells. K562 Cells were double-stained with Annexin V-FITC and PI, and then analyzed by flow cytometry; (B) Effects of M. integrifolia on cell morphology and nucleus of K562 cells. Cells treated for 24, 48 and 72 h were stained with Hoechst 33342. Morphological changes were observed under fluorescent microscope. All experiments were done independently in triplicate per experimental point, and representative results were shown. The results represented the mean ± S.D. of three independent experiments. ** p < 0.01 indicated statistically significant differences vs. Control; (C) Effects of M. integrifolia on the expression of some key apoptotic proteins in K562 cells. K562 cells were treated with M. integrifolia for 24 h. Western blot analysis was performed in triplicate per experimental point; β-actin was used as reference control.

Figure 4

The alterations of mitochondria triggered by M. integrifolia. (A) Effect of M. integrifolia on mitochondrial membrane potential of K562 cells. Cells were treated with M. integrifolia for 5, 10 and 24 h. Then the cells were labeled with Rhodamine 123 and analyzed by flow cytometry. Histograms show number of cell channel (vertical axis) vs. Rhodamine 123 fluorescence (horizontal axis). ** p < 0.01 vs. Control. (Error bars = S.D., n = 3); (B) Detection of release of cytochrome C from mitochondria in K562 cells after M. integrifolia treatment.

Figure 5

ROS generation induced by M. integrifolia. K562 cells were treated with M. intergrifolia for 3, 6 and 9 h. The intracellular ROS level was observed under fluorescent microscope.