Baicalein mediates inhibition of migration and invasiveness of skin carcinoma through Ezrin in A431 cells
Authors: Yu Chen
Ji Li
Hongfu Xie
Bin Wu
Faqing Tang
Weiwei Wang
Youxiang Liao
Xiaowei Tang
Damao Huang
Journal: BMC Cancer (2011)
Discipline: CLINICAL MEDICINE
Keywords: Ezrin; skin cancer; metastasis; baicalein; anti-metastasis therapy
Affiliations: Department of Dermatology, Xiangnan College, Chenzhou 423000, PR China.

Abstract:  Background: Ezrin is highly expressed in skin cancer and promotes tumor metastasis. Ezrin serves as a promising target for anti-metastasis therapy. The aim of this study is to determine if the flavonoid bacailein inhibits the metastasis of skin cancer cells through Ezrin. Methods: Cells from a cutaneous squamous carcinoma cell line, A431, were treated with baicalein at 0-60 μM to establish the non-cytotoxic concentration (NCC) range for baicalein. Following treatment with baicalein within this range, total Ezrin protein (both phosphorylated and unphosphorylated forms) and phosphorylated-Ezrin (phos-Ezrin) were detected by western blotting, and Ezrin RNA was detected in A431 cells using reverse transcription-polymerase chain reaction (RT-PCR). Thereafter, the motility and invasiveness of A431 cells following baicalein treatment were determined using wound-healing and Boyden chamber invasion assays. Short-interfering RNA (si-RNA) specifically targeting Ezrin was transfected into A431 cells, and a si-RNA Ezrin-A431 cell line was established by G418 selection. This stable cell line was transiently transfected with Ezrin and mutant Ezrin plasmids, and its motility and invasiveness was subsequently determined to clarify whether bacailein inhibits these processes through Ezrin. Results: We determined the range of NCCs for baicalein to be 2.5-40 μM in A431 cells. Baicalein displayed a dose- and time-dependent inhibition of expressions of total Ezrin and phos-Ezrin within this range NCCs. In addition, it exerted this inhibitory effect through the reduction of Ezrin RNA transcript. Baicalein also inhibited the motility and invasiveness of A431 skin carcinoma cells within the range of NCCs, in a dose- and time-dependent manner. A431 cell motility and invasiveness were inhibited by 73% and 80% respectively when cells were treated with 20 μM baicalein. However, the motility and invasiveness of A431 cells containing the Ezrin mutant were not effectively inhibited by baicalein. Conclusions: Baicalein reduces the migration and invasiveness of A431 cells through the inhibition of Ezrin expression, which leads to the suppression of tumor metastasis. 

Retracted: Yes

PubPeer comments:
 Fig 5A:  Inhibitory effects of baicalein on the motility and invasiveness of A431 cells. A431 cells were treated with 20 u03bcM bacailein, and cell invasiveness was detected using Boyden Chamber with Matrigel coating. A-(a), Invasiveness of control cells (treated with 0.1% DMSO). A-(b), Invasiveness of cells treated with 20 u03bcM baicalein. ![file] For comparison, Fig 5A from [Huang et al (2011)](http: xbyxb.csu.edu.cn xbwk fileup PDF 201107616.pdf).  Effect of berberine on invasion and mobility of 5-8F cells. A: Invasion cells permeating Boden chamber membrane in the negative control . The similar panel is the negative control in both cases, but A431 cells do not seem to be synonymous with the 5-8F cell-line. ![file] , 
 Fig 2(a,b).  The inhibitory effects of baicalein on Ezrin and phos-Ezrin in A431 cells. a. A431 cells were treated with baicalein at 0, 10, 20, and 40 u03bcM for 24 h. b. A431 cells were treated with 20 u03bcM baicalein for 12, 24, and 48 h. ![file] Fig 6 from Huang et al (2011). I have concerns. ![file] , 
 As a comment to the above comment by Hoya Camphorifolia, note that the following picture appeared in: 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, which has comments [https: pubpeer.com publications 5A01EBABF68C7ECD61B488169888FE] ![file] , 
 #1 In case the link doesn t work, Huang et al (2011) is  [Berberine inhibits the invasion and metastasis of nasopharyngeal carcinoma cells through Ezrin phosphorylation]  . , 
 After increasing brightness of Figure 3A, some repeated background areas seem to be visible. *** ![file] , 
 Retracted [15 March 2022] . ------------------- The Editors have retracted this Article. After publication, concerns were raised regarding the data presented in Figs. 2, 3 and 5. Specifically: - Fig. 2a and b appear highly similar to Fig. 6a in [1] and Fig. 4c in [2]. - Fig. 3a appears to contain straight line breaks and duplicated patterns in the background of the gel. - Fig. 5a appears highly similar to Fig. 5a of [1] representing different cell lines. Thufeffe authors provided the raw data to address these concerns, but the images contained further overlap with [1]. The Editors therefore no longer have confidence in the presented data. Author Faqing Tang has not specifically stated whether they agree with the retraction. Authors Weiwei Wang, Yu Chen and Hongfu Xie have not responded to any correspondence from the Editor or Publisher about this retraction. The Publisher has not been able to obtain current email addresses for authors Bin Wu, Ji Li and Damao Huang. - [1] Huang, D., Wang, W., et al. Berberine inhibits the invasion and metastasis of nasopharyngeal carcinoma cells through Ezrin phosphorylation. J. Cent. South Univ. (Med. Sci.) 36,7, 616-23 (2011). https: doi.org 10.3969 j.issn.1672-7347.2011.07.006 - [2] Tang, F., Wang, D., et al. Berberine inhibits metastasis of nasopharyngeal carcinoma 5-8F cells by targeting Rho kinase-mediated Ezrin phosphorylation at threonine 567. J. Biol. Chem., 284(40), 27456-27466 (2009). https: doi.org 10.1074 jbc.M109.033795 ,