Berberine inhibits metastasis of nasopharyngeal carcinoma 5-8F cells by targeting Rho kinase-mediated Ezrin phosphorylation at threonine 567

Abstract

Ezrin is highly expressed in metastatic tumors and is involved in filopodia formation as well as promotion of tumor metastasis. Thus, Ezrin may serve as a potential target for anti-metastatic therapy. This study demonstrates that berberine reduces filopodia formation of a nasopharyngeal carcinoma (NPC) cell line, 5-8F, at non-cytotoxic concentrations. Furthermore, invasion and motility of 5-8F cells are decreased in a dose- and time-dependent manner, resulting in 73.0% invasion and 67.0% motility inhibition at 20 mum. The inhibitory effects of berberine on 5-8F cell metastasis were further confirmed in a mouse model of metastasis. Berberine treatment in vivo resulted in a 51.1% inhibition of tumor metastasis to the lymph nodes and decreased Ezrin phosphorylation at threonine 567 in metastatic samples. Berberine suppressed the presence of phosphorylated Ezrin (phospho-Ezrin) in a dose- and time-dependent manner but had no effect on total Ezrin protein expression at non-cytotoxic concentrations. Furthermore, the inhibitory effects of berberine on phospho-Ezrin were dependent on the suppression of Rho kinase activity. Reduction of Ezrin phosphorylation at Thr(567) by berberine was associated with its inhibitory effect on filopodia formation in 5-8F cells. However, berberine did not effectively inhibit the motility and invasion of NPC cells containing Ezrin Thr(567) mutants. These results confirm that berberine inhibits Ezrin phosphorylation at Thr(567). Nonetheless, berberine reduces motility and invasion of cells and inhibits tumor metastasis. The reduction of Rho kinase-mediated Ezrin phosphorylation mediated by berberine may be a novel anti-metastatic pathway in NPC 5-8F cells.

FIGURE 1.

Cytotoxic effects of berberine on 5-8F cells. A, structure of berberine, an isoquinoline derivative alkaloid. B, cells were treated with berberine at 2.5, 5, 10, 20, 40, 80, or 100 μm for 24 or 48 h before MTT assay for cell viability. C, after berberine treatment for 48 h, an LDH assay was performed. Data are represented as mean ± S.D. from three independent experiments and statistically analyzed with Student's t test (*, #, p < 0.05; **, ##, p < 0.01). U, units; L, liter.

FIGURE 2.

Inhibitory effects of berberine on 5-8F cell filopodia formation and Ezrin expression. Cells were treated with 40 μm berberine for 48 h. A, cells were stained with fluorescein isothiocyanate-phalloidin and treated with 0.1% DMSO (a), 40 μm berberine (b), and 4′,6-diamidino-2-phenylindole stain (c). d, average number of cells with filopodia were counted from 10 fields. Scale bar, 20 μm. B, cells were observed by scanning electron microscopy after treatment with 0.1% DMSO (a) or 40 μm berberine (b). c, cells with filopodia were counted. Scale bar, 5 μm; arrows point to filopodia. C, Ezrin and phospho-Ezrin expression in 5-8F cells with or without berberine. Cells treated with 0.1% DMSO served as the blank control, and Ezrin and phospho-Ezrin expression were determined by immunoblot. β-Actin served as the loading control.

FIGURE 3.

Berberine inhibition of metastasis in 5-8F cells in vitro and in vivo. In the cell motility and invasion assays, 5-8F cells were treated with berberine at 2.5, 5, 10, or 20 μm for 24 h for dose course assay. Cells were treated with berberine at 5 μm for 12, 24, and 48 h for the time course assay. The treated cells were then assayed for invasion (A) and motility (B), as described. C, invasion of 5-8F cells at various time points. D, motility of 5-8F cells at various time points. Results were analyzed using one-way analysis of variance with post hoc Dunnett's test (*, p < 0.05; **, p < 0.01). In animal experiments, 30 nude BALB/c mice were injected with 5-8F cell suspensions containing Matrigel through the tail vein, 1 × 10⁴ cells/mouse, 10 mice/group. One group treated with the same volume of water served as the control, and two groups were treated with berberine at 15 and 30 mg/kg for 30 days starting on day 1. E, the metastatic tumors from mediastinal lymph node were weighed. F, body weight of the control group and the groups receiving berberine (two-sided Welch's t test; **, p < 0.01).

FIGURE 4.

Ezrin and phospho-Ezrin expression in metastatic tumors and 5-8F cells with or without berberine treatment. A, Ezrin and phospho-Ezrin expression were detected in metastatic tumor samples from the nude mice using immunochemistry. Paraffin sections were stained with hematoxylin and eosin and detected with antibodies against Ezrin or phospho-Ezrin. Sections stained with normal mouse IgG served as the negative control. Arrows, positive cells. Original magnification was ×400. Scale bar, 5 μm. In the in vitro experiments, assays of berberine inhibition on phospho-Ezrin, 5-8F cells were treated with berberine at 2.5, 5, and 10 μm for 24 h for the dose course assay (B), and 5-8F cells were treated with berberine at 5 μm for 12, 24, and 48 h for the time course assay (C) and then subjected to immunoblotting. Three independent experiments were carried out, abundance ratio to β-actin was counted, and data are represented as mean ± S.D. from three experiments. *, p < 0.05. H&E, hematoxylin and eosin.

FIGURE 5.

Berberine inhibits Ezrin phosphorylation through Rho kinase. A, GST-Ezrin phosphorylation mediated by Rho kinase, PKC, GRK2, MRCK, or LOK was assayed in 5-8F cells with berberine treatment as described. B, Rho kinase activity was assayed in 5-8F cells with or without berberine treatment by an in vitro kinase assay; expression of Rho, Ezrin, and phospho-Ezrin was detected using immunoblotting (IB). C, detection of Rho, Rac, or Cdc42 expression by immunoblot is shown in 5-8F cells with or without berberine treatment. D, cells were treated with berberine at 20 or 40 μm for 48 h. Active Rho, Rac, or Cdc42 were precipitated with GST-Rhotekin-RBD and GST-PAK-PBD and were detected with immunoblot using antibodies against Rho, Rac, or Cdc42, respectively. Coomassie Blue staining is shown as a loading control. Abundance ratios to β-actin or GST-Ezrin were calculated, and data are represented as mean ± S.D. from three independent experiments. *, p < 0.05. IP, immunoprecipitation.

FIGURE 6.

Berberine-suppressed Ezrin phosphorylation can be reversed by active Rho kinase. Berberine-treated 5-8F cells were transfected with pcDNA3.1-Rho. Rho was then precipitated with an anti-Rho antibody (IP). GST-Ezrin was phosphorylated by the precipitated Rho kinase using an in vitro kinase assay. Phospho-Ezrin was detected by immunoblotting (IB). GST-Ezrin stained with Coomassie Blue served as the loading control. Three independent experiments were carried out, and densitometric analysis was performed. Abundance ratios to β-actin or GST-Ezrin were calculated, and data are represented as mean ± S.D. from three independent experiments. *, p < 0.05.

FIGURE 7.

Berberine decreases filopodia formation through inhibition of Ezrin phosphorylation. A, Ezrin and phospho-Ezrin in 6-10B cells were detected using immunoblotting. Positive control was untreated 5-8F cells. 6-10B cells were transfected with pcDNA3.1-Ezrin, pcDNA3.1-Ezrin-M(A), or pcDNA3.1. The stably transfected cell lines containing pcDNA3.1-Ezrin, pcDNA3.1-Ezrin-M(A), or pcDNA3.1 were constructed using G418 selection. B, expression of Ezrin and phospho-Ezrin in these stably transfected cell lines was detected by immunoblotting. C, filopodia formation in 6-10B-pcDNA3.1 (a), 6-10B-pcDNA3.1-Ezrin (b), and 6-10B- pcDNA3.1-Ezrin treated with 5 μm berberine (c). d, cells with filopodia were counted. Arrows, filopodia; scale bar, 5 μm.

FIGURE 8.

Berberine inhibits motility and invasion in 6-10B cells through inhibition of phosphorylation of Ezrin at Thr⁵⁶⁷. A, motility (A) and invasion (B) of the stably transfected cell lines 6-10B-pcDNA3.1, 6-10B-pcDNA3.1-Ezrin, 6-10B-pcDNA3.1-Ezrin-M(A), and 6-10B-pcDNA3.1-Ezrin-M(D) with or without berberine treatment. C, stably transfected cells were injected into nude mice that were treated with 15 mg/kg berberine for 30 days. The metastasized tumors in the mediastinal lymph nodes were collected as described. Data are represented as mean ± S.D. from three independent experiments. Results were analyzed by one-way analysis of variance with post hoc Dunnett's test (*, p < 0.05). D, Ezrin and phospho-Ezrin expression from stably transfected cells with or without berberine treatment from three independent experiments. Densitometric analysis was performed, and abundance ratios to β-actin were calculated. Data represent the mean ± S.D. from three independent experiments.

FIGURE 9.

Berberine inhibits motility and invasion in 5-8F-si-Ezrin cells through inhibition of Ezrin phosphorylation at Thr⁵⁶⁷. A, 5-8F cells were transfected with pU6pro-si-Ezrin, and the 5-8F-si-Ezrin cell line was established by G418 selection. Ezrin expression was effectively blocked in these cells. B, detection of motility and invasion of 5-8F-si-Ezrin cells. Motility (C) and invasion (D) from 5-8F-si-Ezrin cells transiently transfected with pcDNA3.1, pcDNA3.1-Ezrin-M(A), pcDNA3.1-Ezrin-M(D), or pcDNA3.1-Ezrin with or without berberine treatment. *, p < 0.05.

FIGURE 10.

Schematic illustration of berberine-inhibited metastasis. Berberine-mediated repression of Ezrin phosphorylation at Thr⁵⁶⁷ through suppression of Rho kinase activity inhibits filopodia formation, resulting in the inhibition of motility and invasion, which leads to decreased metastasis of NPC 5-8F cells.