The in vitro and in vivo antitumor effects of clotrimazole on oral squamous cell carcinoma
Authors: Juan Wang
Yun Hong
Xiaobing Chen
Zirong Kuang
Tong Wu
Lihua Jia
Juan Xia
Bin Cheng
W Keung Leung
Journal: PloS one (2014)
Discipline: Multidisciplinary
Keywords: Clotrimazole; Antitumor effect; Oral squamous cell carcinoma; Apoptosis; Cell cycle arrest
Affiliations: Department of Oral Medicine, Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China., Oral Diagnosis and Polyclinics, Prince Philip Dental Hospital, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China.

Abstract:  Background: Clotrimazole is an antifungal imidazole derivative showing anti- neoplastic effect in some tumors, but its anticancer potential is still unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the antitumor effect of clotrimazole, and to investigate the possible mechanism of clotrimazole-mediated antitumor activity in OSCC. Methodology: In vitro experiments, the cell viability and clonogenic ability of three human OSCC cell lines CAL27, SCC25 and UM1 were detected after clotrimazole treatment by CCK8 assay and colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the involvement of several mediators of apoptosis was examined by western blot analysis. Then, the in vivo antitumor effect of clotrimazole was investigated in CAL27 xenograft model. Immunohistochemistry and western blot analysis were performed to determine the presence of apoptotic cells and the expression of Bcl-2 and Bax in tumors from mice treated with or without clotrimazole. Results: Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Clotrimazole caused cell cycle arrest at the G0/G1 phase. In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax. Notably, clotrimazole treatment inhibited OSCC tumor growth and cell proliferation in CAL27 xenograft model. Clotrimazole also markedly reduced Bcl-2 expression and increased the protein level of Bax in tumor tissues of xenograft model. Conclusion: Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC. 

Retracted: No

PubPeer comments:
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 Expression of Concern, April 2020. https: journals.plos.org plosone article?id=10.1371 journal.pone.0231686 It reads: After publication of this article [1], concerns were raised regarding duplication of well images within Fig 2, and the representation of western blot loading controls in Fig 5. Specifically, In Fig 2A, the CAL27 0u00b5M well image is incorrect, and shows a duplicate of the SCC25 0u00b5M well image In Fig 2A, the SCC25 30u00b5M well image is incorrect and shows a duplicate of the CAL27 30u00b5M well image. Fig 5A and 5B incorrectly show a shared a-tubulin loading control for Bax and Bcl-2 for each experiment. Samples were run on separate gels for Bax and Bcl-2, but the loading controls for the Bcl-2 blots are not shown. The authors have stated there were errors in the selection of images for the published Fig 2A. Further explanation of the methodology is provided by the authors as follows: In the cell colony formation assay, the OSCC cells were seeded into 6-well plates and treated with different concentrations of clotrimazole after 24 h. At the end of the experiment, the colonies consisting of 50 cells were counted under a microscope. All experiments were repeated in triplicate. Images were captured of each well in two of the experiments. Editorial assessment of underlying images for available replicates identified further similarities between published well images and replicate well images labelled as different cell lines. As such, concerns about the accuracy of the image records cannot be fully resolved. The quantitative data underlying the charts in Fig 2B is provided here as S1 File. The available underlying western blot images for Fig 5 are provided in S2 File, including the a-tubulin loading control for the Bcl-2 blot for Fig 5A UM1 cells. The corresponding a-tubulin loading controls for the Bcl-2 blots for Fig 5A CAL27 cells and for Fig 5B are not available. Additionally, the underlying Bax western blots for Fig 5A are not available. Some experimental replicate blots and their associated quantification data are also provided in S2 File. The underlying data for the other figures in the article are not available. The PLOS ONE Editors issue this Expression of Concern to alert readers to the concerns about Fig 2A, and the inaccurate representation of the western blots in Fig 5. ,