[Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells]

Abstract

Objective: To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism.

Methods: The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells.

Results: Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G₀/G₁ (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01).

Conclusion: Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

Keywords: Akt; LY294002; acute myeloid leukemia; apoptotic; cell cycle; matrine.

1

Mat和联合LY294002对K562细胞增殖抑制作用 EInhibitory effect of matrine alone and in combination with LY294002 on proliferation of K562 cells. ^**P < 0.01, compared with the single drug group, the combined medication group of the same time of action.

2

Mat联用LY294002对K562细胞形态的影响 Morphological changes of K562 cells treated with matrine alone or in combination with LY294002. A: Cell morphology observed under inverted microscope (Original magnification: × 200). B: Cell morphology observed with Wright staining under optical microscope (× 1000). C: Cell morphology observed with acridine orange (AO) staining under fluorescence microscope (×200). 1: Control group; 2: Matrine group; 3: LY294002 group; 4: Matrine +LY294002 group.

3

Mat联合LY294002对K562细胞凋亡的影响 Effects of matrine combined with LY294002 on apoptosis of K562 cells detected by flow cytometry. ^**P < 0.01, ^***P < 0.0001. LY: LY294002

4

流式细胞仪检测药物作用48 h后K562细胞的周期分布 Cell cycle distributions detected by flow cytometry after treatment for 48 h with matrine alone or in combination with LY294002.

5

药物作用48 h后对K562细胞周期蛋白和细胞凋亡蛋白表达水平 Expression levels of cyclins and apoptotic proteins in K562 cells after treatment for 48 h with matrine alone or in combination with LY294002 detected by Western blotting. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs control group; ^#P < 0.01, ^##P < 0.001 vs Mat group and LY294002 group. LY: LY294002.

6

药物作用48 h后对K562细胞PI3K/AKT通路相关蛋白表达水平的影响 Expression level of proteins of PI3K/AKT pathway in K562 cells after treatment for 48 h with matrine alone or in combination with LY294002 detected by Western blotting. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs control group; ^#P < 0.01, ^##P < 0.001 vs Mat group and LY294002 group.