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.
Highlights
Ezrin, a member of the ERM family of cytoskeletal proteins, has been implicated in dynamic membranebased processes, such as the formation and stabilization of filopodia [1]
This study demonstrates that berberine reduces filopodia formation of a nasopharyngeal carcinoma (NPC) cell line, 5-8F, at non-cytotoxic concentrations
Berberine significantly inhibits the spontaneous mediastinal lymph node metastasis produced by orthotopic implantation of Lewis lung carcinoma into the lung parenchyma in vivo [27] and inhibits the motility and invasion of highly metastatic A549 cells at non-cytotoxic concentrations in vitro [33]
Summary
Reagents and Antibodies—Berberine was purchased from Sigma. The compound was stored at 4 °C protected from exposure to light. After washing with PBS, the sections were incubated sequentially with a secondary antibody against mouse, peroxidase enzyme label, and diaminobenzidine (Sigma) and stained with hematoxylin (Polysciences, Inc., Warrington, PA), dehydrated, and mounted under a glass coverslip. When cultures were at 70 – 80% confluence, the cells were treated with berberine for 48 h in RPMI 1640 medium containing 10% fetal bovine serum. Treated cells were washed once with ice-cold PBS and lysed in 250 l of kinase lysis buffer (25 mM Tris-HCl (pH 7.5), 5 mM -glycerophosphate, 0.1 mM Na3VO4, 10 mM MgCl2, 1 mM aprotinin, and 1 mM phenylmethanesulfonyl fluoride). A DNA fragment encoding the GTPase-binding domain of p21-activated kinase (PAK-PBD), comprising amino acids 68 –166, was generated by PCR and cloned into the BamHI/XhoI sites of pGEX-5x-1 and expressed in Escherichia coli as GST-PAKPBD fusion protein according to the manufacturer’s protocol. GST fusion proteins were collected by incubation with glutathione-Sepha-
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