Berbamine Inhibits Cell Proliferation and Migration and Induces Cell Death of Lung Cancer Cells via Regulating c-Maf, PI3K/Akt, and MDM2-P53 Pathways

Abstract

Berbamine (BBM) is a natural product isolated from Berberis amurensis Rupr. We investigated the influence of BBM on the cell viability, proliferation, and migration of lung cancer cells and explored the possible mechanisms. The cell viability and proliferation of lung cancer cells were evaluated by MTT assay, EdU assay, and colony formation assay. Migration and invasion abilities of cancer cells were determined through wound scratch assay and Transwell assay. Cell death was evaluated by cell death staining assay and ELISA. The expressions of proteins were evaluated using western blot assay. A xenograft mouse model derived from non-small-cell lung cancer cells was used to detect the effect of BBM on tumor growth and metastasis in vivo. Both colony formation and EdU assays results revealed that BBM (10 μM) significantly inhibited the proliferation of A549 cells (P < 0.001). BBM (10 μM) also significantly inhibited the migration and invasion ability of cancer cells in wound scratch and Transwell assays. Trypan blue assay and ELISA revealed that BBM (20 μM) significantly induced cell death of A549 cells. In xenograft mouse models, the tumor volume was significantly smaller in mice treated with BBM (20 mg/kg). The western blotting assay showed that BBM inhibited the PI3K/Akt and MDM2-p53 signaling pathways, and BBM downregulated the expression of c-Maf. Our results show that BBM inhibits proliferation and metastasis and induces cell death of lung cancer cells in vitro and in vivo. These effects may be achieved by BBM reducing the expression of c-Maf and regulating the PI3K/Akt and MDM2-p53 pathways.

Figure 1

BBM inhibits the cell viability and proliferation of lung cancer cells. Cells were treated with vehicle or various concentrations of BBM, and MTT assays (a, b) (bar = 100 μm in (a)), colony formation assays (c, d) (bar = 100 μm in (c)), and EdU assays (e, f) (bar = 100 μm in (e)) were performed. Data are expressed as mean ± SD (n = 3). ^∗P < 0.05 and ^∗∗∗P < 0.001 vs. Ctrl group.

Figure 2

BBM induces cell death of lung cancer cells. Cells were untreated (Ctrl) or treated with BBM. Cell death was detected through Trypan blue assays (a, b) (the arrows indicated that the cells were stained with Trypan blue and bar = 100 μm in (a)) and Cell Death Detection ELISA (c). Data are expressed as mean ± SD (n = 3). ^∗P < 0.05 and ^∗∗∗P < 0.001 vs. Ctrl group.

Figure 3

BBM inhibits the migration and invasion of lung cancer cells in vitro. Cells were treated with vehicle or various concentrations of BBM. Cell migration and invasion activities were tested by wound scratch assays (a, b), Transwell assays (c, d), and Transwell assays with Matrigel (c, e). Data are expressed as mean ± SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. Ctrl group.

Figure 4

BBM disrupts the PI3K/Akt and MDM2/p53 signal pathways in lung cancer cells. Cells were untreated (Ctrl) or treated with BBM. The expressions of PI3K (a, b), MDM2 (a, c), p-AKT/AKT (a, d), p53 (a, e), c-Maf (a, f), Bcl-2/Bax (a, g), and cleaved-caspase-3/caspase-3 (a, h) were tested by western blot. Data are expressed as mean ± SD (n = 3). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. Ctrl group.

Figure 5

Effects of LY294002 on the cell viability and metastasis of lung cancer cells. Cells were untreated (Ctrl) or treated with LY294002 and BBM. Cell viability was evaluated by MTT assay (a). Metastasis of lung cancer cells was evaluated by wound scratch assay (b) (bar = 100 μm). ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. Ctrl group.

Figure 6

Effects of AMG232 on the cell viability and metastasis of lung cancer cells. Cells were untreated (Ctrl) or treated with AMG232 or BBM. Cell viability was evaluated by MTT assay (a). Metastasis of lung cancer cells was evaluated by wound scratch assay (b) (bar = 100 μm). ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. Ctrl group, and #P < 0.05 vs. AMG232 group.

Figure 7

BBM inhibits tumor growth and metastasis in vivo. Nude mice were implanted with A549 cells. When the tumors reached 150 mm³, the mice were treated with vehicle (Ctrl) or BBM (a). The body weight of the mice (b) and the tumor volume (c) were recorded. Lung weight (e), tumor weight (f), and the foci of metastasis to lung (g) were measured. Data are expressed as mean ± SD (n = 6). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. Ctrl group.

Figure 8

The histopathological changes of lung, liver, kidney, and heart were stained with hematoxylin/eosin and photographed (bar = 100 μm).

Figure 9

BBM disrupts the PI3K/Akt and MDM2/p53 signal pathways in tumors. ALB/c mice were untreated (Ctrl) or treated with BBM; the expressions of PI3K (a, b), MDM2 (a, c), p-Akt/Akt (a, d), p53 (a, e), c-Maf (a, f), Bcl-2/Bax (a, g), and cleaved-caspase-3/caspase-3 (a, h) were examined by western blot (n = 6). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. Ctrl group.

Figure 10

A schematic of the proposed signaling pathway of the current study. BBM downregulates PI3K and Akt and then downregulates the expressions of c-Maf and MDM2, and c-Maf downregulates the expression of MDM2 and eventually upregulates the expression of p53, leading to cell death.