[Matrine inhibits proliferation and promotes autophagy and apoptosis in non-small cell lung cancer cells by deactivating PI3K/AKT/mTOR pathway]

Abstract

Objective: To investigate the inhibitory effect of matrine on the proliferation of human non-small cell lung cancer (NSCLC) and explore the possible molecular mechanism.

Methods: Cultured human NSCLC A549 cells were treated with 0.4, 0.8, 1.2, 1.6, and 2.0 g/L matrine for 24, 48 or 72 h. CCK-8 assay was used for measuring the changes in A549 cell viability. The morphological changes of the cells were observed under a fluorescence microscope, and flow cytometry was employed for analyzing the cell apoptosis. The effects of matrine and the PI3K specific inhibitor LY294002 (10 nmol/L) on AKT pathway and autophagy-related proteins in A549 cells were investigated using Western blotting.

Results: Matrine significantly inhibited the proliferation of A549 cells in a time- and dose-dependent manner (P < 0.05). At the concentration of 1.6 g/L or higher, matrine caused obvious cell shrinkage and fragmentation and significantly increased floating cells; autophagy vacuoles could be observed in the cells after acridine orange staining. Within the concentrations range of 0.8-1.6 g/L, matrine time- and dosedependently increased the cell apoptosis. Treatment of the cells with 1.6 g/L matrine and 10 nmol/L LY294002 resulted in significantly lowered expressions of p-AKT and p-mTOR proteins and increased the expression of light chain 3B (LC 3B), an autophagy-related protein, as compared with those in the control cells (P < 0.05).

Conclusions: We demonstrate that matrine inhibits the proliferation and induces autophagy and apoptosis of A549 cells by deactivating AKT pathway, suggesting the potential of matrine as an anti-cancer agent for lung cancer.

Keywords: AKT pathway; apoptosis; autophagy; matrine; non-small cell lung cancer.

1

苦参碱诱导A549细胞自噬 Matrine induces autophagy in A549 cells. Autophagic vacuoles (red staining) can be seen in A549 cells following treatment with various concentrations of matrine (Acridine orange staining, magnification, ×200).

2

苦参碱对A549细胞的增殖抑制作用呈剂量和时间依赖性 Matrine dose- and time-dependently inhibits A549 cell proliferation. A: Cell viability assessed by CCK-8 assay at 24, 48, and 72 h; B: Inhibition rate of cell proliferation (%). The experiments were performed in triplicate and the data are expressed as Mean±SD (^*P < 0.05).

3

苦参碱诱导A549细胞凋亡，凋亡呈剂量依赖性 Matrine dose-dependently induces apoptosis in A549 cells. The apoptosis of A549 cells treated with 0, 0.8, 1.2, and 1.6 g/L matrine for 48 h was analyzed by flow cytometry with Annexin V/PI staining.

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苦参碱诱导A549细胞凋亡呈时间依赖性 Matrine time-dependently induces apoptosis in A549 cells. Flow cytometry with Annexin V/PI staining showed the apoptotic cell populations after treatment with 1.2 g/L matrine at different time points.

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Western blotting分析显示苦参碱处理后A549细胞中LC 3B升高，p-AKT/p-mTOR降低 Western blotting showing significant upregulation of light chain 3B (LC 3B) expression and reduction of p-AKT and p-mTOR expressions in matrine-treated A549 cells (^*P < 0.05). No significant changes are found in AKT and mTOR protein expressions in A549 cells after matrine treatment.