Epithelial-to-mesenchymal transition markers to predict response of Berberine in suppressing lung cancer invasion and metastasis

Abstract

Background: The effects of berberine on the metastatic potential of lung cancer cells and its underlying mechanisms have not been fully elucidated. Since epithelial-to-mesenchymal transition is a cellular process associated with cancer invasion and metastasis, we attempted to investigate the potential use of berberine as an inhibitor of TGF-β1-induced epithelial-to-mesenchymal in A549 cells.

Methods: In this study, we investigated the anticancer activity of berberine against A549 cells in vitro and in vivo. BBR-induced apoptosis of the human lung cancer cells was determined by flow cytometry. The ability of BBR to inhibit TGF-β-induced EMT was examined by QRT-PCR and Western blotting. The impact of BBR on A549 cell migration and invasion was evaluated by transwell assay.

Results: We demonstrated that TGF-β1 induced epithelial-to-mesenchymal to promote lung cancer invasion and metastasis. Berberine inhibited invasion and migration of A549 cells, increased expression of the epithelial phenotype marker E-cadherin, repressed the expression of the mesenchymal phenotype marker Vimentin, as well as decreased the level of epithelial-to-mesenchymal -inducing transcription factors Snail1 and Slug during the initiation of TGF-β1-induced epithelial-to-mesenchymal. Furthermore, berberine inhibited growth of lung cancer cells in vivo xenograft.

Conclusions: Our findings provided new evidence that berberine is an effective inhibitor of the metastatic potential of A549 cells through suppression of TGF-β1-induced epithelial-to-mesenchymal.

Figure 1

The effect of BBR on cell viability in A549 cells. (A) Chemical structure of berberine hydrochloride. A549 cells (B) and nomal human bronchial epithelial cells (C) were treated with 0, 20, 40, 80 and 160 μM BBR for 48h and 72h. Cell viability was measured using MTT assay. Values are expressed as mean±SD of three experiments. *P<0.05, **P<0.01, as compared with control.

Figure 2

Effect of BBR on cell apoptosis in A549 cells. A549 cells were treated with 0, 20, 40 and 80 μM BBR for 6 h (A), 12 h (B) and 24 h (C). Apoptotic rates were measured using flow cytometry (D). Values are expressed as mean ± SD of three experiments. *P <0.05, **P < 0.01, as compared with control.

Figure 3

BBR inhibits TGF- β1-induced EMT development. (A) Control A549 cells treated with DMSO display classical epithelial morphology (control group); A549 cells show a pebble-like shape and tight cell-cell adhesion. (B) Morphological changes of A549 cells induced by 5 ng/mL of TGF-β1 for 48 h; TGF-β1-treated cells show a decrease in cell-cell contacts and adopt a more elongated morphological shape representing a mesenchymal phenotype (TGF-β group). (C) A549 cells treated with 5 ng/mL of TGF-β1 plus 10 μM of BBR for 48 h also show the mesenchymal phenotype (TGF-β + BBR 10 group). (D) A549 cells treated with 5 ng/mL of TGF-β1 plus 20 μM of BBR for 48 h display epithelial morphology (TGF-β + BBR 20 group) (Magnification × 200).

Figure 4

BBR inhibits TGF-β1-induced EMT markers and transcription factors. (A) Protein level of E-cadherin was measured by Western blotting assay after A549 cells were treated with different concentrations of BBR for 48 h. (B) Protein level of Snail1and Slug were measured by Western blotting assay after A549 cells were treated with different concentration of BBR for 48 h. (C) Protein level of MMP2, MMP-9, p-Smad2/3 and Smad2/3 were measured by Western blotting assay after A549 cells were treated with different concentration of BBR for 48 h. (D) mRNA level of E-cadherin and Vimentin was measured by real-time PCR assay after A549 cells were treated with 20 μM BBR for 24 h. Values are expressed as mean ± SD of three experiments.*P <0.05, **P <0.01, as compared with control group or TGF-β1 stimulation group.

Figure 5

Effect of BBR on lung cancer cell migration and invasion induced by TGF-β1. (A) Effects of BBR on lung cancer cell migration after crystal violet staining by Matrigel migration assay as described (100×). (B) Effects of BBR on lung cancer invasion after crystal violet staining by Matrigel migration assay as described (100×). (C) Matrigel migration of A549 cells counted in five random views. (D) Matrigel invasion of A549 cells counted in five random views. Three independent experiments were performed, *P < 0.05, **P < 0.01.

Figure 6

Inhibition of tumor growth in nude mice xenografted with human A549 cells by BBR. Mice were randomly divided into three groups, five to six mice each. In treated mice, BBR was administered i.p. at a dose of 5 mg/kg or 10 mg/kg three times per week. A significant reduction in tumor volume was observed in treated mice. The figures show the relative tumor volume (A, B) and tumor weight (C).