Berberine inhibits metastasis of nasopharyngeal carcinoma 5-8F cells by targeting Rho kinase-mediated Ezrin phosphorylation at threonine 567
Authors: Lei Wang
Zhiwei He
Daofa Tian
Ya Cao
Dongsheng Wang
Yuan Wu
Feng Zhu
Jingjing Meng
Damao Huang
Chunlei Xie
Yu Chen
Fuliang Deng
Chaojun Duan
Bin Wu
Shujin Liu
Faqing Tang
Weiwei Wang
Journal: Journal of Biological Chemistry (2009)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Ezrin; berberine; metastasis; filopodia formation; anti-metastatic therapy
Affiliations: Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China. 

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. 

Retracted: No

PubPeer comments:
 Six of the picture in Figure 4 are duplicates, appearing in other articles with overlapping authors. One of them appears in as many as four different articles.  Berberine Inhibits Metastasis of Nasopharyngeal Carcinoma 5-8F Cells by Targeting Rho Kinase-mediated Ezrin Phosphorylation at Threonine 567, J Biol Chem. 2009 Oct 2; 284(40): 27456u2013-27466, doi:u00a010.1074 jbc.M109.033795, Figure 4  Identification of novel signaling components in N,Nu2019-Dinitrosopiperazine-mediated metastasis of nasopharyngeal Carcinoma by quantitative phosphoproteomics, BMC Cancer 2014, 14:243, doi:u00a010.1186 1471-2407-14-243, Figure 8  MNAT1 is overexpressed in colorectal cancer and mediates p53 ubiquitindegradation to promote colorectal cancer malignance, J Exp Clin Cancer Res. 2018; 37:284. doi:u00a010.1186 s13046-018-0956-3, figure 8  Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis, Int. J. Mol. Sci. 2014 Nov; 15(11), 20054--20071., doi:u00a010.3390 ijms151120054, Figure 6 , 
 If you were wondering about the remaining two pictures in said figure, then wonder no longer. They are also duplicates, but with a different set of articles. Still overlapping authors.  Berberine Inhibits Metastasis of Nasopharyngeal Carcinoma 5-8F Cells by Targeting Rho Kinase-mediated Ezrin Phosphorylation at Threonine 567, J Biol Chem. 2009 Oct 2; 284(40): 27456u2013-27466, doi:u00a010.1074 jbc.M109.033795, Figure 4  Dinitrosopiperazine-decreased PKP3 through upregulating miR-149 participates in nasopharyngeal carcinoma metastasis, Mol Carcinog. 2018 Dec, 57(12): 1763u2013-1779, doi:u00a010.1002 mc.22895, Figure 8  N,Nu0001-Dinitrosopiperazine-mediated Ezrin Protein Phosphorylation via Activation of Rho Kinase and Protein Kinase C Is Involved in Metastasis of Nasopharyngeal Carcinoma 6-10B Cells, J Biol Chem. 2011 Oct 2; 286(42): 36956--36967, doi:u00a010.1074 jbc.M111.259234, Figure 5 , 
 There are images in Figure 2 and in Figure 7 which also appears in a second article, though they have been modified.  Berberine Inhibits Metastasis of Nasopharyngeal Carcinoma 5-8F Cells by Targeting Rho Kinase-mediated Ezrin Phosphorylation at Threonine 567, J Biol Chem. 2009 Oct 2; 284(40): 27456u2013-27466, doi:u00a010.1074 jbc.M109.033795, Figure 2  Berberine Inhibits Metastasis of Nasopharyngeal Carcinoma 5-8F Cells by Targeting Rho Kinase-mediated Ezrin Phosphorylation at Threonine 567, J Biol Chem. 2009 Oct 2; 284(40): 27456u2013-27466, doi:u00a010.1074 jbc.M109.033795, Figure 7  N,Nu0001-Dinitrosopiperazine-mediated Ezrin Protein Phosphorylation via Activation of Rho Kinase and Protein Kinase C Is Involved in Metastasis of Nasopharyngeal Carcinoma 6-10B Cells, J Biol Chem. 2011 Oct 2; 286(42): 36956--36967, doi:u00a010.1074 jbc.M111.259234 , 
 Some of the western blots also appear in multiple publications. Here is one group of blots which appear in five different articles in different constellations and with modifications.  Berberine Inhibits Metastasis of Nasopharyngeal Carcinoma 5-8F Cells by Targeting Rho Kinase-mediated Ezrin Phosphorylation at Threonine 567, J Biol Chem. 2009 Oct 2; 284(40): 27456u2013-27466., doi:u00a010.1074 jbc.M109.033795, Figure 4  Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis, Int. J. Mol. Sci. 2014 Nov; 15(11), 20054--20071., doi:u00a010.3390 ijms151120054, Figure 5  N,N9-Dinitrosopiperazine-Mediated AGR2 Is Involved in Metastasis of Nasopharyngeal Carcinoma, PLoS ONE. 2014; 9(4): e92081., doi:u00a010.1371 journal.pone.0092081, Figure 5  Baicalein mediates inhibition of migration and invasiveness of skin carcinoma through Ezrin in A431 cells, BMC cancer. 2011 Dec 28; 11:527., doi: 10.1186 1471-2407-11-527., Figure 2   Berberine inhibits the invasion and metastasis of nasopharyngeal carcinoma cells through Ezrin phosphorylation, Zhong Nan Da Xue Bao Yi Xue Ban. 2011 Jul; 36(7):616--23, doi: 10.3969 j.issn.1672-7347.2011.07.006., Figures 5 and 7 ,